Noncoding RNAs and Bone 9811624011, 9789811624018

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Airong Qian · Ye Tian   Editors

Noncoding RNAs and Bone

Noncoding RNAs and Bone

Airong Qian • Ye Tian Editors

Noncoding RNAs and Bone

Editors Airong Qian School of Life Sciences Northwestern Polytechnical University Xi’an, China

Ye Tian School of Life Sciences Northwestern Polytechnical University Xi’an, China

ISBN 978-981-16-2401-8 ISBN 978-981-16-2402-5 https://doi.org/10.1007/978-981-16-2402-5

(eBook)

© Springer Nature Singapore Pte Ltd. 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

We feel exciting and honored to produce a book on nocoding RNAs (ncRNAs) and bone. In our lab, we focus on ncRNAs and various bone diseases especially osteoporosis and got some research achievements about ncRNAs and different types of osteoporosis after years of study. In this book, the aim is to provide an outline of the function, molecular mechanism, and application of ncRNAs in various bone diseases. Bone, as an endocrine organ and principal structural connective tissue, serves roles in maintaining mineral homeostasis and energy metabolism, supporting locomotion, and protecting important organs. Bone cells are categorized into five types: (1) osteoblasts, which build new bone tissue; (2) osteoclasts, which break down bone tissue; (3) osteocytes, which live as long as the organism itself and hold the bone together; (4) lining cells, which cover non-remodeling bone surfaces and protect the bone; (5) chondrocytes, which produce and maintain the cartilaginous matrix. A series of serious bone diseases including osteoporosis, osteoarthritis, and osteosarcoma are induced by functional imbalance of these cells. In the past few years, ncRNAs have been proved to play an essential role in controlling target gene expression underlying various processes of bone cells, thus dysregulation of ncRNAs is involved in the pathogenesis and progression of bone diseases. NcRNAs are endogenous RNA transcripts that lack conserved open reading frames (ORFs) and have no capability to be translated into proteins and play a role in the transcription or post-transcriptional period. It is estimated that more than 90% of the human genome undergoes transcription, while only 2% codes for proteins. According to the size of nucleotides, ncRNAs can be mainly classified as miRNA (microRNA, 200 nt). MiRNAs are frequently regarded as epigenetic modifier, functioning in a series of cell activities such as cell activation, proliferation, differentiation, and self-renewal by binding to the complementary sequence of 3’ untranslated region (3’ UTR) of target genes, resulting in the degradation of target mRNA or translation inhibition. LncRNAs

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were considered to participate in a series of biological processes such as transcription, translation, epigenetic regulation, splicing, chromosome dosage compensation, imprinting, nuclear and cytoplasmic trafficking, and cell cycle control. Studies have shown that the abnormal expression of ncRNA is closely related to the process of occurrence and progression of bone-related diseases. Emerging evidences have proved the indispensable roles of ncRNAs in homeostasis of skeleton system. The book provides an in-depth and comprehensive overview of the essential role of ncRNAs in bone formation. In combination with researches from multiple scholars in this field, the book reviews the mechanisms of ncRNA-related bone diseases as well as the potential applications of RNA synthesis technology in bone disorder treatments. This volume covers the following topics: (1) basic introduction of ncRNAs and bone development, how (2) microRNAs and (3) LncRNAs regulate the bone formation, (4) how ncRNAs and the corresponding pathways participate in bone metabolism diseases, and (5) RNA synthesis technology and the possible RNA therapies in bone disease. Researchers and students in the fields of human genetics, human physiology, developmental biology, and biomedical engineering, as well as professionals and scientists in orthopedics, will particularly find this book helpful. As an editor, I hope that the book could meet the reader’s expectations and I am grateful to all the authors for their excellent contribution to the book. I would also like to acknowledge those who helped make this book possible: the Associate Editor, Ye Tian, for her tireless and fastidious dedication to the mission and professional support; my cooperators, Ge Zhang and Chao Liang at the Hong Kong Baptist University, Hui Li at the Xi’an Hong-hui Hospital, Xue Wang, Ying Huai, Zhihao Chen, Dijie Li, Chong Yin, Qian Huang, Shenxing Tan, Jiawei Pei, Peihong Su, Mili Ji, and Xiaohua Chu at Northwestern Polytechnical University, for their constant indispensable support and constructive suggestions. A special thanks is addressed to the Springer editors for their perfect and professional service in completing the editing. Without their contributions, this book would never have come into existence. This work was supported by the Natural Science Foundation of China (82072106, 31570940, 31370845, 81772017, 31400725, 81700784, 32071517, 32000924, 81901917, and 81801871), the China Postdoctoral Science Foundation (2020M683573, 2019T120947, 2018T111099, 2017M610653, 2017M613196, 2017M613210, 2017M623249, 2015T81051, and 2014M562450), the New Century Excellent Talents in University (NCET-12-0469), the Shenzhen Science and Technology Project (JCYJ20160229174320053), the Fundamental Research Funds for the Central Universities (3102019ghxm012, 3102018zy053, 3102017OQD041, 3102017OQD050, 3102016ZY037, and 3102014JKY15007), Young Talent Fund of University Association for Science and Technology in Shaanxi, China (20170401), the Project Supported by Natural Science Basic Research Plan in Shaanxi Province of China (2021JQ-128, 2020JM-100, 2018JM3040, 2018SF263, 2018KA180038C180038, 2018JQ8032, 2018JQ3049, 2015JM3078, and 2015JQ3076), Shaanxi Provincial Key R&D Program (2018KWZ-10), Shaanxi

Preface

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Postdoctoral Science Foundation (2017BSHEDZZ13), Special Fund for Technological Innovation of Shaanxi Province (No. 2019QYPY-207), and the Innovation Foundation for Doctor Dissertation of Northwestern Polytechnical University (CX201821). Xi'an‚ China

Airong Qian Ye Tian

Contents

Part I

MicroRNAs and Bone

1

MicroRNAs and Osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xue Wang, Ruiyun Li, Xuechao Liang, Ye Tian, Airong Qian, and Hui Li

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MicroRNAs and the Diagnosis of Osteoporosis . . . . . . . . . . . . . . . . . Ying Huai, Hui Li, Ye Tian, Airong Qian, and Zhihao Chen

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MicroRNAs and Osteoarthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shenxing Tan, Qian Huang, Xuechao Liang, Airong Qian, and Ye Tian

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Mechanosensitive MicroRNAs and Bone Formation . . . . . . . . . . . . . Zhihao Chen, Yan Zhang, Ying Huai, Fan Zhao, Lifang Hu, Chaofei Yang, Ye Tian, and Airong Qian

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Part II

Long Noncoding RNAs and Bone

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Roles and Mechanism of Long Noncoding RNAs in Bone Diseases . . Dijie Li, Chaofei Yang, Ye Tian, Zhihao Chen, Airong Qian, and Chong Yin

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Long Noncoding RNAs Regulate Osteoblast Function and Bone Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Chong Yin, Ye Tian, Xuechao Liang, Dijie Li, Shanfeng Jiang, Xue Wang, and Airong Qian

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Contents

Part III

RNA Synthesis Technology and RNA Therapy in Bone Diseases

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Synthetic Technology of Noncoding RNAs Used in Bone Disease Research and Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Ye Tian, Chong Yin, Chaofei Yang, Mili Ji, Xiaohua Chu, and Airong Qian

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RNA Therapy in Bone Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Jiawei Pei, Qian Huang, Mili Ji, Xiaohua Chu, Ye Tian, Airong Qian, and Peihong Su

Part I

MicroRNAs and Bone

Chapter 1

MicroRNAs and Osteoporosis Xue Wang, Ruiyun Li, Xuechao Liang, Ye Tian, Airong Qian, and Hui Li

Contents 1.1 1.2 1.3 1.4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MiRNAs Involved in the Regulation of Osteoblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MiRNAs Involved in the Regulation of Osteoclasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MiRNAs Involved in the Regulation of Bone-Related Signaling Pathways . . . . . . . . . . . . . . 1.4.1 MiRNAs Involved in the Regulation of Wnt Signaling Pathway . . . . . . . . . . . . . . . . . 1.4.2 MiRNAs Involved in the Regulation of BMP Signaling Pathway . . . . . . . . . . . . . . . . 1.4.3 MiRNAs Involved in the Regulation of TGF-β Signaling Pathway . . . . . . . . . . . . . . 1.4.4 MiRNAs Involved in the Regulation of RANK/RANKL/ OPG Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.5 MiRNAs Involved in the Regulation of M-CSF/c-FMS Signaling Pathway . . . . . 1.5 Conclusion and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5 7 9 11 11 16 17 18 19 20 20

Abstract Osteoporosis is one of the most common bone disorders, characterized by low bone mass and deterioration of the bone tissue, and seriously affects the health and quality of life of elderly individuals and postmenopausal women. Small microRNAs (miRNAs) are emerging as epigenetic regulators of gene expression which abnormal expression is closely related to the process of occurrence and progression of bone-related diseases, especially osteoporosis. Nowadays, it is widely accepted that miRNAs exert crucial roles in the regulation of osteoporosis by modulating osteoblast and osteoclast function and targeting multiple signaling pathways. However, the concrete pathogenesis of miRNAs on osteoporosis has

X. Wang · R. Li · X. Liang · Y. Tian · A. Qian Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, School of Life Sciences, Northwestern Polytechnical University, Xi’an, China e-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected] H. Li (*) Department of Adult Joint Reconstruction, Xi’an Hong-Hui Hospital, Xi’an, China © Springer Nature Singapore Pte Ltd. 2021 A. Qian, Y. Tian (eds.), Noncoding RNAs and Bone, https://doi.org/10.1007/978-981-16-2402-5_1

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not been fully elucidated yet. This chapter aims to provide an updated overview of recent advances in the roles of miRNAs on osteoporosis, focusing on the interaction between miRNAs and specific molecules in the process of osteoblastogenesis and osteoclastogenesis as well as cascade reaction implicated in bone-related signaling pathways. Keywords MiRNAs · Osteoporosis · Osteoblasts · Osteoclasts · Bone-related signaling pathways

Abbreviation 30 UTR ALK ALP AP-1 APC Bgn BMMs BMPs BMSC CDC42 c-FMS CTSK CXCL12 DC-STAMP DKK1 DKK2 GSK3β hADSCs hBMSCs HMGA2 HMOBs hMSCs JNK LEF LRP MAPK M-CSF MITF MMP MNGC MSCs NFATC

30 untranslated region Activin receptor-like kinase Alkaline phosphatase Activator protein 1 Adenomatous polyposis coli Biglycan Bone marrow monocytes Bone morphogenetic proteins Bone mensencymal stem cell Cell division cycle 42 Colony-stimulating factor-1 receptor Cathepsin K C-X-C motif chemokine ligand 12 Dendritic cell-specific transmembrane protein Dickkopf-1 Dickkopf-2 Glycogen synthase kinase 3β Human adipose-derived mesenchymal stem cells Human bone mesenchymal stem cells High mobility group AT-Hook 2 Human mandibular osteoblast-like cells Human mesenchymal stem cells c-Jun N-terminal kinase lymphoid enhancer-binding factor1 Low density lipoprotein receptor-related protein Mitogen-activated protein kinase Macrophage-colony-stimulating factor Microphthalmia-associated transcription factor Matrix metalloproteinase Multinucleated giant cells Mesenchymal stem cells Nuclear factor of activated T cells C

1 MicroRNAs and Osteoporosis

NFATc1 OCN OPG OPN OSX PDCD4 PGC-1α PGE2 PMOP PPAR-gamma PRKACB PTEN PVDF RANKL rBMSCs RTK Runx2 SCD-1 sFRP SIRT6 Smad Sox6 Spry1 SPRY2 TCF TGFBRI TGF-β TNFSF13b Tob1 TRAF3 TRAF6 TRAP Wnt XPO5

1.1

5

Nuclear factor of activated T cells 1 Osteocalcin Osteoprotegerin Osteopontin Osterix Programmed cell death 4 Progastricsin 1α Prostaglandin E2 Postmenopausal osteoporosis Peroxisome proliferator activated receptor gamma Protein kinase A catalytic subunit B Phosphatase and tensin homolog Poly (vinylidene-trifluoroethylene)/barium titanate Receptor activator of nuclear factor Kappa-B ligand Rat bone mesenchymal stem cells Receptor tyrosine kinase Runt-related transcription factor 2 Stearoyl-CoA desaturase 1 Secreted frizzled related protein Sirtuin6 SMA- and MAD-related protein SRY-box transcription factor 6 Sprouty 1 Sprouty 2 T cell-specific transcription factor Transforming growth factor-β receptor I Transforming growth factor-beta Tumor necrosis factor superfamily member 13b Transducer of RbeB2 receptor tyrosine kinase 1 Tumor necrosis factor receptor associated factor-3 Tumor necrosis factor receptor associated factor-6 Tartrate-resistant acid phosphatase Wingless-type MMTV integration site family members Exportin 5

Introduction

Osteoporosis is a common and chronic disease with deterioration of microarchitecture, reduction in bone strength, and increase of bone fragility, leading to higher risk of bone fracture, which seriously affect both sexes and all races, exerting a strong influence on life quality, morbidity, and even mortality [1]. Emerging evidence suggests that it is a kind of multifactorial and complicated metabolic

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osteopathy, in which many kinds of cells in the bone microenvironment, especially osteoblast and osteoclast, playing critical roles and coordinating the bone homeostasis [2]. The remodeling of bone is mainly maintained by osteoblast-medicated bone formation and osteoclast-medicated bone resorption. A disequilibrium between bone formation and bone resorption can result in metabolic bone diseases [2]. It is reported that genetic factors, behaviors (the level of physical activity) as well as nutrients (calcium intake) are recognized be critical determinants of osteoporosis [3]. Epigenetic modification was proposed to describe the interaction between gene and environmental factors during development, regulating and determining the ultimate fate of tissues and organs. Accumulating evidence suggests that epigenetic modification plays an important role in the plasticity of phenotypes under environmental factors, which may be one of the underlying mechanism of increased risks of osteoporosis [4]. It is particularly important to elucidate the epigenetic mechanism operative in the development of osteoporosis. MicroRNAs (miRNAs) are well-known as the most abundant regulatory noncoding RNAs that play significant roles in the regulation of gene expression through transcriptional and post-transcriptional regulation by binding to the complementary sequence of 30 untranslated regions (3’-UTR) of target genes, resulting in the degradation of target mRNA or translation inhibition. Mature miRNAs are generated by the sequential cleavage of precursor transcripts. Initially, miRNA coding genes are transcripted into ~1000 nt primary miRNA (pri-miRNA) in nucleus, which is subsequently processed by microprocessor complex composed of DROSHA and DGCR8 as precursor miRNA (pre-miRNA), ~70 nt nucleotides with stem-loop structure, then transported to cytoplasm by Exportin 5 (XPO5). In the cytoplasm, the pre-miRNA is further processed by DICER, producing a duplex RNA of 22 nt with its 30 ends having a two-nucleotide overhang. Subsequently, a miRNA duplex is loaded onto one of the Ago family proteins, together with several auxiliary proteins from the GW182 family, to form the RNA-induced silencing complex (RISC) [5, 6] (Fig. 1.1). Because of its role in regulating and modifying other RNAs, regulatory miRNAs are frequently regarded as epigenetic modifier, functioning in a series of cell activities such as cell activation, proliferation, differentiation, and self-renewal. Mounting evidence suggests that the deregulation of miRNAs is closely related to the occurrence and development of osteoporosis [7]. Currently, researches show that miRNAs have been deeply involved in the regulation of bone remodeling and mineralization [8]. Osteoblast and osteoclast, two main cell populations in bone homeostasis, are responsible for bone formation and bone resorption, respectively, which phenotypic differentiation and growth have been largely investigated be controlled by miRNAs. Additionally, in vivo and in vitro evidence have established that aberrant expression of miRNAs occurs in osteoporosis samples, through influencing bone functions and several microenvironment signals such as Wingless-type MMTV Integration Site Family Members (Wnt), transforming growth factor-beta (TGF-β), Bone Morphogenetic Proteins (BMPs), and Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL)/Osteoprotegerin (OPG) ratio, involving in the regulation of multiple bone-related signaling pathways.

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Fig. 1.1 The schematic of biosynthesis of miRNA. In nucleic, miRNA coding gene are transcripted into pri-miRNA by RNA polymerase II, then processed by Drosha/DGCR8 as pre-miRNA which subsequently will be exported to the cytoplasm. In the cytoplasm, pre-miRNA is further cleaved by the endonuclease Dicer to mature miRNA which loads in RISC to suppress translation or degrade target mRNA

High or low expression miRNAs target to transcription factors or significant signaling molecules, making it possible to destroy the balance between bone formation and bone resorption, and leading to the occurrence of osteoporosis. However, the exact mechanism by which miRNA regulates the occurrence and development of osteoporosis has not been demonstrated yet. In this chapter, we mainly focused on the emerging roles of miRNAs in osteoporosis development and emphasized recent advances in understanding regulation of miRNAs on osteoblast, osteoclast, and bone-related signaling pathways.

1.2

MiRNAs Involved in the Regulation of Osteoblasts

Osteoblasts, originate from the pluripotent mesenchymal stem cells (MSCs), are responsible for the synthesis, secretion, and mineralization of bone matrix. As the main functional cells for bone formation, osteoblasts express different critical bone-

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related marker genes at the different stages of differentiation, such as alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osterix (OSX), osteocalcin (OCN), which is an important activity in the maintains of bone homeostasis and microstructure. Numerous studies have shown that dysfunction of osteoblasts is one of decisive factors incurring osteoporosis. The roles of miRNAs in the regulation of osteoblastic function have been largely investigated. Several miRNAs are implicated in the proliferation of osteoblast, involving in the regulation of osteoporosis. MiR-23a was reported to inhibit the proliferation and differentiation of osteoblast via targeting progastricsin 1α (PGC-1α) in osteoporosis rat induced by tretinoin [9]. Exosomal miR-150-3p promotes osteoblast proliferation and differentiation in osteoporosis [10]. MiR-342 represses MC3T3-E1 cell proliferation, migration, and differentiation [11]. MiR-122 exerts inhibitory effect on both proliferation and differentiation of osteoblasts in ovariectomized rats with osteoporosis [12]. In addition, Li et al. reported BMSC-derived exosomes carrying microRNA-122-5p was implicated in the regulation of osteoblasts, in which miR-122-5p negatively regulated Sprouty 2 (SPRY2) and elevated the activity of receptor tyrosine kinase (RTK), thereby promoting the proliferation and differentiation of osteoblasts [13]. Besides, overexpression of miR-186 inhibits osteoblast differentiation in human bone mesenchymal stem cells (hBMSCs) by targeting Sirtuin6 (SIRT6), which has been reported to mediate osteogenic differentiation in rat bone mesenchymal stem cells (rBMSCs) [14]. Several miRNAs such as miR-374b, miR-208, miR-138 exert positive or negative function in regulating osteoblast differentiation [15–17]. In particular, Chen et al. showed how the tail vein injection of antagomir of miR-138, a mechano-sensitive miRNA, in hindlimb unloading mice model bring partial rescue of osteoporosis [17]. MiRNAs that modulate or target osteogenic pivotal marker genes, specific molecules, and common regulated factors (Alp, Ocn, β-catenin, Runx2, Osx, etc.) play significant roles in the regulation of osteoblasts. MiR-497-5p targets high mobility group AT-Hook 2 (HMGA2), promoting mineralized nodule formation and the expression of Runx2 and OCN [18]. The study of Suman et al. showed that overexpression of miR-300 in the rat calvarial osteoblasts decreases the protein levels of SMA- and MAD-related protein (Smad3), β-catenin, and Runx2. MiR-300 intervenes Smad3/β-catenin/RunX2 crosstalk, negatively regulating osteoblasts differentiation, which unveil an enormous ability to serve as a therapeutic target for bone-related disorder management strategies [19]. OSX is known as an essential transcription factor in osteoblasts and osteocytes, exerting pivotal role in matrix mineralization and bone formation [20]. MiR-485-5p was reported to restrain cell viability and the expression level of osteogenic markers. Further study showed that OSX is a direct target of miR-485-5p [21]. The inhibition of miR-608 promotes the expression of Runx2 and OSX in osteoblasts [22]. Moreover, SRY-box transcription factor 6 (Sox6) plays a role in the regulation of osteoblast differentiation, confirmed as a target of miR-17-3p [23]. Additionally, miR-130b overexpression or inhibition significantly promoted or suppressed osteogenic differentiation of osteoblasts, respectively, by directly targeting phosphatase and tensin homolog (PTEN) in osteoblast [24]. MiR-199a

1 MicroRNAs and Osteoporosis

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can rescue ALP activity and osteoblast differentiation via repressing Klotho protein and messenger RNA expression, affecting the downstream fibroblast growth factor receptor 1/extracellular-signal-regulated kinase and Janus kinase 1/signal transducer and activator of transcription 1 pathways [25].

1.3

MiRNAs Involved in the Regulation of Osteoclasts

Osteoclasts consist of multinucleated giant cells (MNGCs), derived from osteoclast precursors. It mainly distributed on the surface of bone and around the bone vessel channel, responsible for bone resorption [26]. Apart from an osteoblast biology, miRNAs are implicated in the process of osteoclastogenesis, playing significant roles in maintaining bone homeostasis [27, 28]. Researches showed that miRNAs also participate in the regulation of osteoclasts biology, including differentiation, cell–cell fusion, and apoptosis [29]. Furthermore, the expression pattern of miRNAs related with the osteoclast activities acting on the specific targets in macrophagecolony-stimulating factor 1 (M-CSF) and RANKL-mediated signaling pathways have also been explored in the following. MiR-483, miR-125a-5p, and miR-338-3p are involved in the pathogenesis of osteoporosis by promoting osteoclast differentiation [30–32]. On the contrary, miR-101 is an important regulator in bisphosphonates treated-osteoclasts and inhibits osteoclast differentiation. MiR-100-5p inhibits osteoclastogenesis and bone resorption by regulating fibroblast growth factor 21 [33]. Tumor necrosis factor superfamily member 13b (TNFSF13b) was reported to participate in the glucocorticoid-induced osteoclast formation. MiR-29a wards off glucocorticoidmediated excessive bone resorption by repressing the TNFSF13b modulation of osteoclastic activity [34]. Inhibition of osteoclasts formation and bone resorption by estrogen is very important in the etiology of postmenopausal osteoporosis. Xu et al. showed miR-27a decreased osteoclast differentiation and bone resorption, involving in the regulation of estrogen-inhibited osteoclast differentiation and function [35]. These data suggest that miRNAs exert essential roles in the process of osteoclast differentiation and function. Fusion of pre-osteoclasts towards multinucleated osteoclasts is the last step of mature osteoclast formation. Salvador et al. found miR-142-3p had inhibitory effect on the conversion of a third osteoclast precursor cell type-dendritic cells to osteoclasts, as a miRNA that is significantly, differentially expressed throughout osteoclastogenesis during miRNA profiling of monocyte-to-osteoclast differentiation [36]. MiR-7b inhibits osteoclastic dendritic cell-specific transmembrane protein (DC-STAMP) expression, implicated in cell–cell fusion for the formation of mature osteoclasts [37]. It needs to be further explored to illuminate the mechanism of cellular fusion in the process of osteoclast maturation. Osteoclast apoptosis is also a vital event in bone metabolism. MiR-539 was reported to induce apoptosis-related genes such as RhoA, caspase-3, and Bcl-2 expression downregulation in osteoclasts [38]. In addition, the above-mentioned

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miR-142-3p was also identified as a negative regulator of osteoclastogenesis from the 3 main precursor cell types: monocytes, macrophages, and dendritic cells, which might be associated with osteoclast apoptosis [36]. Tumor necrosis factor receptor associated factor-3 (TRAF3) is thought as a regulator of apoptosis, inhibited by miR-346-3p overexpression, suggesting miR-346-3p could be a novel therapeutic target for osteoclast apoptosis- related bone loss [39]. MiRNAs also regulate important molecules which are responsible for modifying matrix composition and liberating soluble factors implicated in osteoclast differentiation in osteoclasts, like matrix metalloproteinase (MMPs) family. MiR-133a targets to MMP9 to regulate the amount of osteoclasts [40]. MiR-126 inhibits osteoclastogenesis by targeting of MMP13 mRNA, a negative regulator of osteoclast differentiation [41]. Beyond that, miRNAs participate in regulating osteoclastspecific genes tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), and nuclear factor of activated T cells cytoplasmic 1 (NFATC) expression. MiR-222-3p serves as an inhibitor of osteoclastogenesis and the inhibition of miR-222-3p upregulates the mRNA levels of NFATc1 and TRAP [42]. MiR-186 regulates CTSK expression, leading to CTSK suppression and decrease of osteoclast activities [43]. Moreover, miR-21, a well-known oncogene, has abnormal expression in many types of cancers, involving in the regulation of bone development, bone remodeling, and bone loss. MiR-21 regulates the bone resorption and osteoclastogenesis under the mechanical force by affecting the cell abilities of proliferation and migration [44]. It has been also reported that miR-21/ mice increased RANKL and decreased OPG through targeting Sprouty 1 (Spry1), however, interestingly, miR-21 deficiency showed increased trabecular bone mass accrual physiologically. Furthermore, miR-21 targets to programmed cell death 4 (PDCD4) mRNA, despite the existence of RANKL, inhibiting bone resorption and osteoclast function [45]. In addition, there is also the latest research showed miR-21 promoted osseointegration and mineralization through enhancing both osteogenic and osteoclastic expression [46]. The research of miR-21 in bone metabolism is worth further exploration. It worths noting that several miRNAs regulate both osteoblasts and osteoclasts, possessing the potential to modulate osteoblasts-osteoclasts crosstalk. A recent study showed that the inhibition of miR-99a simultaneously promote the commitment into osteogenic differentiation, suppress osteoclastogenesis, by reciprocally interfering cellular communication [47]. MiR-29a signaling in osteogenic cells protects bone tissue from osteoporosis through repressing osteoclast regulators RANKL and C-XC motif chemokine ligand 12 (CXCL12) to reduce osteoclastogenic differentiation [48]. The alteration of age-related molecules in the bone marrow microenvironment is one of the driving forces in osteoporosis. Jiang et al. found miR-31a-5p from aging BMSCs links bone formation and resorption in the bone marrow microenvironment, and inhibition of miR-31a-5p prevents bone loss and decreases the osteoclastic activity of aged rats [49]. However, further study of more issues regarding the detailed actions of miRNAs on the interaction between osteoblast and osteoclast is necessary.

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MiRNAs Involved in the Regulation of Bone-Related Signaling Pathways

The role of several miRNAs in the regulation of the occurrence and development of osteoporosis by targeting positive or negative regulators in osteoblast and osteoclastrelated signaling pathways is increasingly evidenced. In this part, the roles of miRNAs acting on the specific targets including vital signaling transduction molecules, regulators, and transcription factors will be discussed. Table 1.1 showed a list of the miRNAs with validated targets in bone-related signaling pathways and Figs. 1.2 and 1.3 showed diagrams of signaling pathway of miRNAs involved in osteoblast and osteoclast.

1.4.1

MiRNAs Involved in the Regulation of Wnt Signaling Pathway

The Wnt pathway regulates a wide variety of cellular processes during embryogenesis and in adult regenerative tissues, which is bound up with bone-related disorders like osteoporosis [50]. It has been well accepted that Wnt signaling cascade activation leads to the promotion of bone formation and suppression of bone resorption, contributing to a balance in bone remodeling. Wnt signaling thus has become a desired target to treat osteoporosis [51]. Wnt canonical signal through frizzled/low density lipoprotein receptor-related protein (LRP)5/6 activation, initiates intracellular signal transduction, interfering the ubiquitinoylation and proteasomal degradation of β-catenin result from the phosphorylation of axin/adenomatous polyposis coli (APC)/glycogen synthase kinase 3β (GSK3β) complex and accumulating cytoplasmic β-catenin level. Then β-catenin is translocated into the nucleus where it interacts with transcription factors such as lymphoid enhancer-binding factor1/T cell-specific transcription factor (LEF/TCF) to activate the transcription of target genes. Whereas non-canonical Wnt5a activation increases intracellular calcium via protein kinase C-dependent mechanisms or induces Rho- or c-Jun N-terminal kinase-dependent changes in the actin cytoskeleton, meanwhile, which will affect the Runx2-related bone formation and peroxisome proliferator activated receptor gamma (PPAR-γ)medicated adipogenesis [52–54]. MiRNA acts as a key regulator involved in Wnt signaling pathway through targeting positive or negative molecules to regulate onset and development of osteoporosis [55]. They negatively regulate translation of specific target mRNAs by base pairing with partially or fully complementary sequences in Wnt signaling pathway. The disruption of the pathway caused by miRNA targeting to Wnt ligand or receptor lead to the attenuation of osteoblast differentiation. For instance, miR-376c targets Wnt-3 and suppresses the binding of Wnt-3 to Fzd and LRP5/6 receptors which prevents the release of β-catenin and its transactivation thereby inhibiting osteoblast differentiation [56]. Similarly, miR-22-3p and miR-34a-5p

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Table 1.1 The roles of miRNAs involved in different bone-related signaling pathways

Signaling pathways Wnt signaling pathway

BMP signaling pathway

TGF-β signaling pathway

miRNA miR-376c miR-223p miR-34a5p miR-3753p miR-5453p miR-4739 miR-27a miR-26b miR-139 miR-217 miR-4833p miR-128 miR-96

Target Wnt-3 Wnt-1

LRP5

Cell line/animal model Calvarial osteoblast Clinical osteoporosis sample Clinical osteoporosis sample Pre-osteoblasts

Inhibition

LRP5

Pre-osteoblasts

Inhibition

LRP3 APC GSK-3β β-Catenin DKK1 DKK2

hBMSCs Pre-osteoblasts rBMSCs hBMSCs MSCs rBMSCs

Inhibition Promotion Promotion Inhibition Promotion Promotion

DKK2 SOST

rBMSC Ankylosing spondylitis mice/ primary osteoblasts hADSCs Human femoral neck fracture samples hBMSCs MSCs hMSCs hADSCs Pre-myogenic C2C12 cells

Promotion Promotion

Inhibition Inhibition Inhibition Promotion Inhibition

PMOP mice rBMSCs

Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Promotion Promotion Promotion Promotion

TGFβ3

Pre-osteoblasts Pre-osteoblasts mMSCs/miR-185/ mice mBMSC/PMOP mice hADSCs mMSCs BMSCs Col1a1-miR23aC transgenic mice Pre-osteoblasts

RANKL

Osteoclastss

Promotion

Wnt-1

Let-7c miR-93

SCD-1 BMP2

miR-98 miR-106a miR-765 miR-450b miR-494

BMP2 BMP2 BMP6 BMP3 BMPR2, RUNX2 BMPR-II SMAD1

miR-1187 miR-2033p miR-135 miR-155 miR-185 Let-7a-5p miR-10b miR-21 miR-130a miR-23a27a-24-2 miR-1403p miR-34a

Effect on bone formation Inhibition Inhibition

SMAD5 SMAD5 BGN TGFBR1 SMAD2 SMAD7 SMURF2 PRDM16

Inhibition

Inhibition Inhibition

Promotion

(continued)

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Table 1.1 (continued)

Signaling pathways RANK/RANKL/OPG signaling pathway

M-CSF/c-FMS signaling pathway

miRNA miR-212 miR-384 miR302a-3p miR-214 miR-1425p

Target RUNX2 RUNX2 PRKACB

Cell line/animal model hMSCs/PMOP mice hMSCs/PMOP mice HMOBs

Effect on bone formation Inhibition Inhibition Promotion

PTEN PTEN

BMMs BMMs

Inhibition Promotion

Abbreviations in the table are shown in the acronym table

target to Wnt1 mRNA, inhibiting osteogenesis [57]. Besides, miR-375-3p and miR-545-3p negatively regulate osteogenesis by targeting LRP5 [58, 59]. MiR-4739 targets to Wnt receptor LRP3, promoting adipogenic and suppressing osteogenic differentiation of human bone marrow stromal cells [60]. Wnt signaling on outer membrane is blocked by these miRNAs, which result in the inhibition of osteogenesis. In addition, circulating miR-194 was recently reported as inhibitory factor of bone formation in senile osteoporosis and postmenopausal osteoporosis by targeting Wnt5a in non-canonical Wnt signaling pathway. β-catenin, as a critical mediator in the cascade of Wnt signal, plays a decisive role in osteogenic differentiation [61]. MiRNAs which stimulate β-catenin accumulation or inhibit the factors related to β-catenin degradation, promoting bone formation. MiR-27a promotes pre-osteoblasts MC3T3-E1 differentiation to osteoblasts and decreases the expression of its target APC, a negative regulator of β-catenin activation, preventing β-catenin from degradation [62]. Some miRNAs as miR-26b and miR-124 have been recently reported to enhance osteogenesis through direct targeting of glycogen synthase kinase 3β (GSK-3β), inducing β-catenin accumulation and translocating into nucleus [63, 64]. MiR-139 negatively regulates osteogenesis by direct targeting β-catenin [65]. In contrast, there are also several miRNAs that target negative regulators in Wnt signaling pathway to induce osteogenesis. These negative regulators include dikkopf (Dkk), sclerostin (SOST), and secreted frizzled related protein (sFRP) that inhibit Wnt binding co-receptor or bind to Wnt molecules. MiR-291a-3p and miR-217 promote osteoblastic differentiation through targeting Dkk-1, which is a powerful antagonist of canonical Wnt signaling pathway and regarded as a biomarker for osteoporosis [66, 67]. Similarly, miR-483-3p and miR-128 directly and functionally target Dkk-2 in rat bone marrow mesenchyml stem cell, thus promoting osteoblastic differentiation [68, 69]. MiR-96 promotes osteoblast differentiation and bone formation by binding to SOST [70]. Additionally, the expression of miRNAs that target the factor implicated in the lipid modification of Wnt proteins indirectly affect osteogenic differentiation. Stearoyl-CoA Desaturase 1 (SCD-1) has been shown to play a key role in Wnt biogenesis and processing, functioning as a positive regulator of osteogenesis.

Fig. 1.2 Schematic drawing of WNT pathway, BMP pathway, and TGF-β pathway implicated in osteoblast differentiation, in which miRNAs bind to the 3’-UTR of the mRNA of the target protein

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Fig. 1.3 Chematic drawing of RANKL pathway and M-CSF/c-FMS pathway implicated in osteoclast differentiation, in which miRNAs bind to the 3’-UTR of the mRNA of the target protein

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Research shows let-7c inhibits osteogenesis and Wnt/β-catenin signaling activation by targeting to SCD-1 mRNA [71].

1.4.2

MiRNAs Involved in the Regulation of BMP Signaling Pathway

BMP-induced receptor activation signaling pathway triggers classical pathway. The binding of the BMP ligand to its receptor (BMPR) induces auto-phosphorylation of BMPR and the activation of the receptor-specific SMA- and MAD-related protein 1/5/8 (Smad1/5/8) protein to assemble a complex with Smad4, translocating into the nucleus and regulating the transcription of the target genes [72]. Moreover, BMPR activation is cross-linked with many other signaling pathways, including the activation of mitogen-activated protein kinase (MAPK) pathway [73]. It is demonstrated that BMP/Smad signaling plays an important role during bone formation and homeostasis. BMPs, members of the TGF-β superfamily of proteins, are thought to control the commitment of mesenchymal stem cells to the osteoblast phenotype [74, 75]. BMPs have long been identified as important players in bone formation, involving in the regulation of the differentiation of pluripotent mesenchymal stem cells into osteogenic cells. BMP2 can stimulate osteoblast differentiation and enhance their function. MiR-93 and miR-98 target BMP2 mRNA to inhibit the expression of osteogenic key proteins, including OSX, Osteopontin (OPN) and Runx2 in hBMSC [76, 77]. Similarly, miR-106a also targets BMP2, negatively regulating osteogenic differentiation of MSC in vitro [78]. MiR-765 was found binding to the 3’-UTR of BMP6 and reduced Smad1/5/9 phosphorylation, thus suppressing osteoblast differentiation of hBMSCs [79]. BMP3, a suppressor member of the BMP family in the regulation of osteogenesis and bone formation, was identified as a direct target of miR-450b. Downregulation of the endogenous expression of BMP3 could mimic the effect of miR-450b upregulation on the osteogenic differentiation of human adiposederived mesenchymal stem cells (hADSCs), promoting osteogenic differentiation in vitro and enhancing bone formation in vivo [80]. A mechanical responsive miR-494, whose overexpression is correlated with a marked reduction in osteoblast differentiation genes and a decrease in osteogenesis in BMP2-induced osteogenic differentiation. Further studies revealed that miR-494 directly targeted BMPR2 and Runx2, participating in the regulation of BMP/Smad signaling pathway and inhibition of bone formation [81]. Singh et al. reported that novel miR-1187 functions as a negative regulator of osteogenesis by repressing BMPR-II expression, activates cell division cycle 42 (CDC42), a major regulator in actin reorganization, and inhibits actin cytoskeletal rearrangement via suppressing non-Smad BMP2/CDC42 signaling pathway [82]. MiR-203-3p inhibits osteogenesis in the jaws of diabetic rats and in rat bone marrow mesenchyml stem cell cultured in high-glucose medium via targeting the

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Smad1 [83]. Two miRNAs, miR-135 and miR-155, inhibits osteoblast differentiation, through targeting of Smad5 and suppressing the translation of Samd5 in mouse pre-osteoblast cells [84, 85]. Some molecules acting as medium in BMP/Smad cascade can be regulated by miRNAs involved in the development of osteoporosis. In vivo experiment, miR-185 negatively regulated osteogenesis through directly targeting biglycan (Bgn), an extracellular matrix proteoglycan acting as a promoter in Bmp4-induced osteoblast differentiation of murine calvarial cells and highly expressed in bone and skeletal connective tissues, which activates Smad1/5/8 signaling and promotes bone formation through the BMP/Smad signaling pathway [86]. Research showed miR-26a treatment could effectively improve the osteogenic differentiation capability of mesenchymal stem cells isolated from littermate-derived ovariectomized osteoporotic mice both in vitro and in vivo. MiR-26a exerts its effect by directly targeting transducer of RbeB2 receptor tyrosine kinase 1 (Tob1), the negative regulator of BMP/Smad signaling pathway by binding to the 3’-UTR and thus repressing Tob1 protein expression [87]. MiR-26a may be a promising therapeutic candidate to enhance bone formation in treatment of osteoporosis and to promote bone regeneration in osteoporotic fracture healing.

1.4.3

MiRNAs Involved in the Regulation of TGF-β Signaling Pathway

TGF-β signaling pathway functions in regulating growth, proliferation, differentiation and extracellular matrix and remodeling of cells, which abnormal signaling transduction is closely related to osteoporosis [88]. TGF-β ligand binds to a tetrameric complex receptor comprising transforming growth factor-β receptor I (TGFBRI) or activin receptor-like kinase (ALK) and transforming growth factor-β receptor II (TGFBRII), giving rise to phosphorylation and kinase activities of receptors, Smad2/3 activation with Smad4 recruitment, which activate intracellular signaling cascade reaction and induce transcription of different factors [88]. TGFBRI was shown to be a direct target of let-7a-5p, and let-7a-5p might inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells in postmenopausal osteoporosis mice by regulating TGFBRI [89]. MiR-10b, identified as a novel target for controlling bone and metabolic diseases, through directly targeting to Smad2, suppresses adipocytic differentiation and promotes osteogenic differentiation and bone formation in human hADSCs via the TGF-β signaling pathway [90]. Smad7 is known as an antagonist of TGF-β/Smad signaling and negatively regulates Runx2. MiR-21 regulates mouse mesenchymal stem cells differentiation by Smad7 targeting [91]. MiR-130a has been reported on the function of controlling differentiation switch fate of bone marrow mesenchyml stem cell during aging. The study of Liang et al. showed miR-130a depressed Smad regulatory factors 2 (Smurf2), a differentiation repressors of TGF-β pathway, increasing osteogenic

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differentiation of BMSC and attenuating adipogenic differentiation in bone marrow mesenchyml stem cell [92]. Lee et al. reported that the miR-23a cluster (miR-23a27a-24-2) promoted osteoblasts differentiation by modulating the TGF-β signaling pathway through targeting of Prdm16, a negative regulator of the TGF-β pathway [93]. Notably, miRNA which links Wnt pathway with TGF-β pathway, participating in regulating osteoblast differentiation as a critical regulatory factor has also been reported. MiR-140-3p has been evidenced as an important linker between Wnt3a and TGFβ3 in MC3T3-E1 cells, of which Wnt3a overexpression inhibited miR-1403p level but enhanced the expression of TGFβ3. Further study showed that TGFβ3 was a direct target of miR-140-3p, but the exact mechanism underlying which needs deep exploration [94].

1.4.4

MiRNAs Involved in the Regulation of RANK/RANKL/ OPG Signaling Pathway

The RANKL/RANK/OPG signaling pathway mainly regulates osteoclast differentiation and resorption activity, participating in physiological and pathological bone reconstruction, earliest mainly found in the research of bone metabolism, osteoporosis, and other fields [95]. RANKL secreted by osteoblast binds to RANK expressed on osteoclast, allowing tumor necrosis factor receptor associated factor6 (TRAF-6) accumulation in osteoclasts and activating IKK/NF-κB, c-Jun N-terminal kinase (JNK), activator protein 1 (AP-1), c-Myc, c-Fos, and nuclear factor of activated T cells 1 (NFATc1), which promote osteoclasts generation, differentiation, and maturation. The inhibition of the role of osteoclasts has already been the key to inhibiting bone mass loss. OPG was reported as secreted protein which competitively binds RANKL with RANK to block osteoclast progenitor differentiation induced by osteoblasts, thereby inhibiting osteoclast formation [96]. Hence, RANKL/OPG ratio is the critical factor in determining osteoclast differentiation. Almeida et al. recently showed miR-99a as a bidirectional regulator involved in the regulation of bone metabolism. The inhibition of miR-99a-5p increased the Tnfrsf11b (OPG encoding gene)/Tnfsf11 (RANKL encoding gene) mRNA expression ratio, thus increasing secreted OPG level, while overexpression of miR-99a-5p exerted reverse effects that it decreased the number of multinucleated cells and downregulated the expression of osteoclastogenic markers, impairing the osteoclastogenic potential of RAW 264.7 cells, which implicated the significance of miR-99a-5p in intercellular communication between bone formation and bone resorption [47]. The expression of miRNA has direct and indirect effects on RANKL level and then influence osteoclast differentiation. MiR-29a inhibits RANKL secretion in osteoblasts through binding to 3’-UTR of RANKL [48]. MiR-34a has been reported to promote bone formation induced by the poly (vinylidenetrifluoroethylene)/barium titanate (PVDF) and repress osteoclast differentiation

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through targeting RANKL [97]. Interference of miR-212 and miR-384 alleviate osteoporosis and promote bone formation by targeting Runx2. Besides, increased OPG and decreased RANKL were shown after miR-212 and miR-384 inhibition, activating OPG/RANKL pathway, which implied that a regulatory mechanism between these two miRNAs, and OPG/RANKL pathway need to be further explored [98]. Additionally, study showed miR-302a-3p indirectly regulated RANKL expression in human mandibular osteoblast-like cells (hMOBs) under the involvement of prostaglandin E2 (PGE2) which had been shown to stimulate the expression of RANKL, mimicking inflammatory conditions. Further research showed that miR-302a-3p target protein kinase A catalytic subunit B (PRKACB) mRNA in cAMP/PKA pathway, which might suppress PRKACB, leading to downregulate subsequent RANKL level [99]. These miRNAs affect the expression of RANKL involved in the regulation of RANKL/OPG signaling pathway. MiR-340 was downregulated during osteoclast differentiation induced by macrophage-colonystimulating factor (M-CSF) and RANKL. Microphthalmia-associated transcription factor (MITF), served as pivotal transcription factor involved in osteoclast differentiation, was found as a target of miR-340. Overexpression of miR-340 inhibited osteoclast differentiation and suppressed both the mRNA and protein level of MITF [100].

1.4.5

MiRNAs Involved in the Regulation of M-CSF/c-FMS Signaling Pathway

The proliferation and survival of osteoclasts depend on the combination of osteoblast-derived M-CSF and osteoclast receptor colony-stimulating factor-1 receptor (c-FMS) which accompanied by activation of downstream cascade signal including PTEN/PI3K/AKT pathway and NF-κB pathway [101]. MiR-214 plays a critical role in osteoclast differentiation that upregulated miR-214 promotes osteoclastogenesis from bone marrow monocytes (BMMs) through PI3K/Akt pathway by targeting PTEN inducing an upregulation of phosphorylated AKT and NFATc1, after RANKL induction. In vivo results show osteoclast-specific miR-214 transgenic mice exhibit downregulated PTEN levels, increased osteoclast activity, and reduced bone mineral density [102]. PTEN was testified to be a direct target of miR-142-5p. Besides, treatment of LY29004 (an inhibitor of the PI3k/Akt pathway) can attenuate miR-142-5p osteoclastogenesis effects. MiR-142-5p promotes osteoclast differentiation of bone marrow-derived macrophages via activating PTEN/PI3K/AKT/FoxO1 pathway [103].

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Conclusion and Future Perspectives

Osteoporosis is a systemic bone disorder defined by low bone mineral density occurring due to an imbalance of osteoclastic and osteoblastic activities. According to the current therapeutics, the most common way to treat osteoporosis is to prevent further bone loss by medicine administration. However, it is necessary to seek alternative methods to treat osteoporosis, as traditional long-term osteoporosis treatment can cause serious side effects. With the emergence of the relationship between miRNA and osteoporosis, the high efficiency, low toxicity clinical treatment is expecting to be established. In this chapter, we highlight the wide range of microRNAs that participate in the regulation of osteoblast and osteoclast and the pathways, exerting crucial roles in maintaining bone remodeling and bone metabolism, which will provide a deeper insight and understanding of the role of miRNAs in osteoporosis into the early diagnosis and prevention of osteoporosis. It also can provide unique opportunities to develop novel therapeutic approaches of osteoporosis and its related bone fracture. Acknowledgements This work was supported by the Natural Science Foundation of China (82072106, and 81801871), the China Postdoctoral Science Foundation (2020 M683573, 2019 T120947, and 2017 M613210), the New Century Excellent Talents in University (NCET12-0469), the Shenzhen Science and Technology Project (JCYJ20160229174320053), Shaanxi Provincial Key R&D Program (2018KWZ-10), Shaanxi Postdoctoral Science Foundation (2017BSHEDZZ13), Special Fund for Technological Innovation of Shaanxi Province (No. 2019QYPY-207), the Fundamental Research Funds for the Central Universities (3102018zy053).

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41. Wu Z, Yin H, Liu T, Yan W, Li Z, Chen J, Chen H, Wang T, Jiang Z, Zhou W, Xiao J (2014) Mir-126-5p regulates osteoclast differentiation and bone resorption in giant cell tumor through inhibition of mmp-13. Biochem Biophys Res Commun 443(3):944–949. https://doi.org/10. 1016/j.bbrc.2013.12.075 42. Takigawa S, Chen A, Wan Q, Na S, Sudo A, Yokota H, Hamamura K (2016) Role of mir-2223p in c-src-mediated regulation of osteoclastogenesis. Int J Mol Sci 17(2):240. https://doi.org/ 10.3390/ijms17020240 43. Ma Y, Yang H, Huang J (2018) Icariin ameliorates dexamethasoneinduced bone deterioration in an experimental mouse model via activation of microrna186 inhibition of cathepsin k. Mol Med Rep 17(1):1633–1641. https://doi.org/10.3892/mmr.2017.8065 44. Li M, Zhang Z, Gu X, Jin Y, Feng C, Yang S, Wei F (2020) Microrna-21 affects mechanical force-induced midpalatal suture remodelling. Cell Prolif 53(1):e12697. https://doi.org/10. 1111/cpr.12697 45. Hu CH, Sui BD, Du FY, Shuai Y, Zheng CX, Zhao P, Yu XR, Jin Y (2017) Mir-21 deficiency inhibits osteoclast function and prevents bone loss in mice. Sci Rep 7:43191. https://doi.org/ 10.1038/srep43191 46. Geng Z, Yu Y, Li Z, Ma L, Zhu S, Liang Y, Cui Z, Wang J, Yang X, Liu C (2020) Mir-21 promotes osseointegration and mineralization through enhancing both osteogenic and osteoclastic expression. Mater Sci Eng C Mater Biol Appl 111:110785. https://doi.org/10.1016/j. msec.2020.110785 47. Moura SR, Bras JP, Freitas J, Osorio H, Barbosa MA, Santos SG, Almeida MI (2020) Mir-99a in bone homeostasis: regulating osteogenic lineage commitment and osteoclast differentiation. Bone 134:115303. https://doi.org/10.1016/j.bone.2020.115303 48. Lian WS, Ko JY, Chen YS, Ke HJ, Hsieh CK, Kuo CW, Wang SY, Huang BW, Tseng JG, Wang FS (2019) Microrna-29a represses osteoclast formation and protects against osteoporosis by regulating pcaf-mediated rankl and cxcl12. Cell Death Dis 10(10):705. https://doi.org/ 10.1038/s41419-019-1942-1 49. Xu R, Shen X, Si Y, Fu Y, Zhu W, Xiao T, Fu Z, Zhang P, Cheng J, Jiang H (2018) Microrna31a-5p from aging bmscs links bone formation and resorption in the aged bone marrow microenvironment. Aging Cell 17(4):e12794. https://doi.org/10.1111/acel.12794 50. Majidinia M, Aghazadeh J, Jahanban-Esfahlani R, Yousefi B (2018) The roles of wnt/betacatenin pathway in tissue development and regenerative medicine. J Cell Physiol 233 (8):5598–5612. https://doi.org/10.1002/jcp.26265 51. Baron R, Gori F (2018) Targeting wnt signaling in the treatment of osteoporosis. Curr Opin Pharmacol 40:134–141. https://doi.org/10.1016/j.coph.2018.04.011 52. Prgomet Z, Axelsson L, Lindberg P, Andersson T (2015) Migration and invasion of oral squamous carcinoma cells is promoted by wnt5a, a regulator of cancer progression. J Oral Pathol Med 44(10):776–784. https://doi.org/10.1111/jop.12292 53. Yuan Z, Li Q, Luo S, Liu Z, Luo D, Zhang B, Zhang D, Rao P, Xiao J (2016) Ppargamma and wnt signaling in adipogenic and osteogenic differentiation of mesenchymal stem cells. Curr Stem Cell Res Ther 11(3):216–225. https://doi.org/10.2174/1574888x10666150519093429 54. Muruganandan S, Roman AA, Sinal CJ (2009) Adipocyte differentiation of bone marrowderived mesenchymal stem cells: cross talk with the osteoblastogenic program. Cell Mol Life Sci 66(2):236–253. https://doi.org/10.1007/s00018-008-8429-z 55. Karimi Z, Seyedjafari E, Khojasteh A, Hashemi SM, Kazemi B, Mohammadi-Yeganeh S (2020) Microrna-218 competes with differentiation media in the induction of osteogenic differentiation of mesenchymal stem cell by regulating beta-catenin inhibitors. Mol Biol Rep 47(11):8451–8463. https://doi.org/10.1007/s11033-020-05885-7 56. Kureel J, John AA, Prakash R, Singh D (2018) Mir 376c inhibits osteoblastogenesis by targeting wnt3 and arf-gef-1 -facilitated augmentation of beta-catenin transactivation. J Cell Biochem 119(4):3293–3303. https://doi.org/10.1002/jcb.26490

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57. Makitie RE, Hackl M, Niinimaki R, Kakko S, Grillari J, Makitie O (2018) Altered microrna profile in osteoporosis caused by impaired wnt signaling. J Clin Endocrinol Metab 103 (5):1985–1996. https://doi.org/10.1210/jc.2017-02585 58. Sun T, Li CT, Xiong L, Ning Z, Leung F, Peng S, Lu WW (2017) Mir-375-3p negatively regulates osteogenesis by targeting and decreasing the expression levels of lrp5 and betacatenin. PLoS One 12(2):e0171281. https://doi.org/10.1371/journal.pone.0171281 59. Li L, Qiu X, Sun Y, Zhang N, Wang L (2019) Sp1-stimulated mir-545-3p inhibits osteogenesis via targeting lrp5-activated wnt/beta-catenin signaling. Biochem Biophys Res Commun 517 (1):103–110. https://doi.org/10.1016/j.bbrc.2019.07.025 60. Elsafadi M, Manikandan M, Alajez NM, Hamam R, Dawud RA, Aldahmash A, Iqbal Z, Alfayez M, Kassem M, Mahmood A (2017) Microrna-4739 regulates osteogenic and adipocytic differentiation of immortalized human bone marrow stromal cells via targeting lrp3. Stem Cell Res 20:94–104. https://doi.org/10.1016/j.scr.2017.03.001 61. Fan J, Su YW, Hassanshahi M, Fan CM, Peymanfar Y, Piergentili A, Del Bello F, Quaglia W, Xian CJ (2020) Beta-catenin signaling is important for osteogenesis and hematopoiesis recovery following methotrexate chemotherapy in rats. J Cell Physiol 236:3740–3751. https://doi.org/10.1002/jcp.30114 62. Xu Q, Cui Y, Luan J, Zhou X, Li H, Han J (2018) Exosomes from c2c12 myoblasts enhance osteogenic differentiation of mc3t3-e1 pre-osteoblasts by delivering mir-27a-3p. Biochem Biophys Res Commun 498(1):32–37. https://doi.org/10.1016/j.bbrc.2018.02.144 63. Hu H, Zhao C, Zhang P, Liu Y, Jiang Y, Wu E, Xue H, Liu C, Li Z (2019) Mir-26b modulates oa induced bmsc osteogenesis through regulating gsk3beta/beta-catenin pathway. Exp Mol Pathol 107:158–164. https://doi.org/10.1016/j.yexmp.2019.02.003 64. Tang SL, Huang QH, Wu LG, Liu C, Cai AL (2018) Mir-124 regulates osteoblast differentiation through gsk-3beta in ankylosing spondylitis. Eur Rev Med Pharmacol Sci 22 (20):6616–6624. https://doi.org/10.26355/eurrev_201810_16136 65. Long H, Sun B, Cheng L, Zhao S, Zhu Y, Zhao R, Zhu J (2017) Mir-139-5p represses bmsc osteogenesis via targeting wnt/beta-catenin signaling pathway. DNA Cell Biol 36(8):715–724. https://doi.org/10.1089/dna.2017.3657 66. Li ZH, Hu H, Zhang XY, Liu GD, Ran B, Zhang PG, Liao MM, Wu YC (2020) Mir-291a-3p regulates the bmscs differentiation via targeting dkk1 in dexamethasone-induced osteoporosis. Kaohsiung J Med Sci 36(1):35–42. https://doi.org/10.1002/kjm2.12134 67. Dai Z, Jin Y, Zheng J, Liu K, Zhao J, Zhang S, Wu F, Sun Z (2019) Mir-217 promotes cell proliferation and osteogenic differentiation of bmscs by targeting dkk1 in steroid-associated osteonecrosis. Biomed Pharmacother 109:1112–1119. https://doi.org/10.1016/j.biopha.2018. 10.166 68. Wang C, Qiao X, Zhang Z, Li C (2020) Mir-128 promotes osteogenic differentiation of bone marrow mesenchymal stem cells in rat by targeting dkk2. Biosci Rep 40(2):BSR20182121. https://doi.org/10.1042/BSR20182121 69. Zhou B, Peng K, Wang G, Chen W, Liu P, Chen F, Kang Y (2020) Mir4833p promotes the osteogenesis of human osteoblasts by targeting dikkopf 2 (dkk2) and the wnt signaling pathway. Int J Mol Med 46(4):1571–1581. https://doi.org/10.3892/ijmm.2020.4694 70. Ma S, Wang DD, Ma CY, Zhang YD (2019) Microrna-96 promotes osteoblast differentiation and bone formation in ankylosing spondylitis mice through activating the wnt signaling pathway by binding to sost. J Cell Biochem 120(9):15429–15442. https://doi.org/10.1002/ jcb.28810 71. Zhou Z, Lu Y, Wang Y, Du L, Zhang Y, Tao J (2019) Let-7c regulates proliferation and osteodifferentiation of human adipose-derived mesenchymal stem cells under oxidative stress by targeting scd-1. Am J Physiol Cell Physiol 316(1):C57–C69. https://doi.org/10.1152/ ajpcell.00211.2018 72. Liang C, Peng S, Li J, Lu J, Guan D, Jiang F, Lu C, Li F, He X, Zhu H, Au DWT, Yang D, Zhang BT, Lu A, Zhang G (2018) Inhibition of osteoblastic smurf1 promotes bone formation in mouse models of distinctive age-related osteoporosis. Nat Commun 9(1):3428. https://doi. org/10.1038/s41467-018-05974-z

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73. Li Y, Liu Z, Tang Y, Feng W, Zhao C, Liao J, Zhang C, Chen H, Ren Y, Dong S, Liu Y, Hu N, Huang W (2020) Schnurri-3 regulates bmp9-induced osteogenic differentiation and angiogenesis of human amniotic mesenchymal stem cells through runx2 and vegf. Cell Death Dis 11 (1):72. https://doi.org/10.1038/s41419-020-2279-5 74. Acil Y, Ghoniem AA, Wiltfang J, Gierloff M (2014) Optimizing the osteogenic differentiation of human mesenchymal stromal cells by the synergistic action of growth factors. J Craniomaxillofac Surg 42(8):2002–2009. https://doi.org/10.1016/j.jcms.2014.09.006 75. Bhat A, Wooten RM, Jayasuriya AC (2013) Secretion of growth factors from macrophages when cultured with microparticles. J Biomed Mater Res A 101(11):3170–3180. https://doi. org/10.1002/jbm.a.34604 76. Zhang Y, Wei QS, Ding WB, Zhang LL, Wang HC, Zhu YJ, He W, Chai YN, Liu YW (2017) Increased microrna-93-5p inhibits osteogenic differentiation by targeting bone morphogenetic protein-2. PLoS One 12(8):e0182678. https://doi.org/10.1371/journal.pone.0182678 77. Zhang GP, Zhang J, Zhu CH, Lin L, Wang J, Zhang HJ, Li J, Yu XG, Zhao ZS, Dong W, Liu GB (2017) Microrna-98 regulates osteogenic differentiation of human bone mesenchymal stromal cells by targeting bmp2. J Cell Mol Med 21(2):254–264. https://doi.org/10.1111/ jcmm.12961 78. Liu K, Jing Y, Zhang W, Fu X, Zhao H, Zhou X, Tao Y, Yang H, Zhang Y, Zen K, Zhang C, Li D, Shi Q (2017) Silencing mir-106b accelerates osteogenesis of mesenchymal stem cells and rescues against glucocorticoid-induced osteoporosis by targeting bmp2. Bone 97:130–138. https://doi.org/10.1016/j.bone.2017.01.014 79. Wang T, Zhang C, Wu C, Liu J, Yu H, Zhou X, Zhang J, Wang X, He S, Xu X, Ma B, Che X, Li W (2020) Mir-765 inhibits the osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting bmp6 via regulating the bmp6/smad1/5/9 signaling pathway. Stem Cell Res Ther 11(1):62. https://doi.org/10.1186/s13287-020-1579-0 80. Fan L, Fan J, Liu Y, Li T, Xu H, Yang Y, Deng L, Li H, Zhao RC (2018) Mir-450b promotes osteogenic differentiation in vitro and enhances bone formation in vivo by targeting bmp3. Stem Cells Dev 27(9):600–611. https://doi.org/10.1089/scd.2017.0276 81. Qin W, Liu L, Wang Y, Wang Z, Yang A, Wang T (2019) Mir-494 inhibits osteoblast differentiation by regulating bmp signaling in simulated microgravity. Endocrine 65 (2):426–439. https://doi.org/10.1007/s12020-019-01952-7 82. John AA, Prakash R, Kureel J, Singh D (2018) Identification of novel microrna inhibiting actin cytoskeletal rearrangement thereby suppressing osteoblast differentiation. J Mol Med 96 (5):427–444. https://doi.org/10.1007/s00109-018-1624-y 83. Tang Y, Zheng L, Zhou J, Chen Y, Yang L, Deng F, Hu Y (2018) Mir2033p participates in the suppression of diabetesassociated osteogenesis in the jaw bone through targeting smad1. Int J Mol Med 41(3):1595–1607. https://doi.org/10.3892/ijmm.2018.3373 84. Iaculli F, Di Filippo ES, Piattelli A, Mancinelli R, Fulle S (2017) Dental pulp stem cells grown on dental implant titanium surfaces: an in vitro evaluation of differentiation and micrornas expression. J Biomed Mater Res B Appl Biomater 105(5):953–965. https://doi.org/10.1002/ jbm.b.33628 85. Gu Y, Ma L, Song L, Li X, Chen D, Bai X (2017) Mir-155 inhibits mouse osteoblast differentiation by suppressing smad5 expression. Biomed Res Int 2017:1893520. https://doi. org/10.1155/2017/1893520 86. Cui Q, Xing J, Yu M, Wang Y, Xu J, Gu Y, Nan X, Ma W, Liu H, Zhao H (2019) Mmu-mir185 depletion promotes osteogenic differentiation and suppresses bone loss in osteoporosis through the bgn-mediated bmp/smad pathway. Cell Death Dis 10(3):172. https://doi.org/10. 1038/s41419-019-1428-1 87. Li Y, Fan L, Hu J, Zhang L, Liao L, Liu S, Wu D, Yang P, Shen L, Chen J, Jin Y (2015) Mir-26a rescues bone regeneration deficiency of mesenchymal stem cells derived from osteoporotic mice. Mol Ther 23(8):1349–1357. https://doi.org/10.1038/mt.2015.101 88. Aslani S, Abhari A, Sakhinia E, Sanajou D, Rajabi H, Rahimzadeh S (2019) Interplay between micrornas and wnt, transforming growth factor-beta, and bone morphogenic protein signaling

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pathways promote osteoblastic differentiation of mesenchymal stem cells. J Cell Physiol 234 (6):8082–8093. https://doi.org/10.1002/jcp.27582 89. Ma W, Dou Q, Ha X (2019) Let-7a-5p inhibits bmscs osteogenesis in postmenopausal osteoporosis mice. Biochem Biophys Res Commun 510(1):53–58. https://doi.org/10.1016/j. bbrc.2019.01.003 90. Li H, Fan J, Fan L, Li T, Yang Y, Xu H, Deng L, Li J, Li T, Weng X, Wang S, Chunhua Zhao R (2018) Mirna-10b reciprocally stimulates osteogenesis and inhibits adipogenesis partly through the tgf-beta/smad2 signaling pathway. Aging Dis 9(6):1058–1073. https://doi.org/ 10.14336/AD.2018.0214 91. Arumugam B, Balagangadharan K, Selvamurugan N (2018) Syringic acid, a phenolic acid, promotes osteoblast differentiation by stimulation of runx2 expression and targeting of smad7 by mir-21 in mouse mesenchymal stem cells. J Cell Commun Signal 12(3):561–573. https:// doi.org/10.1007/s12079-018-0449-3 92. Lin Z, He H, Wang M, Liang J (2019) Microrna-130a controls bone marrow mesenchymal stem cell differentiation towards the osteoblastic and adipogenic fate. Cell Prolif 52(6):e12688. https://doi.org/10.1111/cpr.12688 93. Zeng HC, Bae Y, Dawson BC, Chen Y, Bertin T, Munivez E, Campeau PM, Tao J, Chen R, Lee BH (2017) Microrna mir-23a cluster promotes osteocyte differentiation by regulating tgf-beta signalling in osteoblasts. Nat Commun 8:15000. https://doi.org/10.1038/ ncomms15000 94. Fushimi S, Nohno T, Nagatsuka H, Katsuyama H (2018) Involvement of mir-140-3p in wnt3a and tgfbeta3 signaling pathways during osteoblast differentiation in mc3t3-e1 cells. Genes Cells 23(7):517–527. https://doi.org/10.1111/gtc.12591 95. Terpos E, Ntanasis-Stathopoulos I (2020) Controversies in the use of new bone-modifying therapies in multiple myeloma. Br J Haematol. https://doi.org/10.1111/bjh.17256 96. Cawley KM, Bustamante-Gomez NC, Guha AG, MacLeod RS, Xiong J, Gubrij I, Liu Y, Mulkey R, Palmieri M, Thostenson JD, Goellner JJ, O'Brien CA (2020) Local production of osteoprotegerin by osteoblasts suppresses bone resorption. Cell Rep 32(10):108052. https:// doi.org/10.1016/j.celrep.2020.108052 97. Lopes HB, Ferraz EP, Almeida AL, Florio P, Gimenes R, Rosa AL, Beloti MM (2016) Participation of microrna-34a and rankl on bone repair induced by poly(vinylidenetrifluoroethylene)/barium titanate membrane. J Biomater Sci Polym Ed 27(13):1369–1379. https://doi.org/10.1080/09205063.2016.1203217 98. Zhang Y, Jiang Y, Luo Y, Zeng Y (2020) Interference of mir-212 and mir-384 promotes osteogenic differentiation via targeting runx2 in osteoporosis. Exp Mol Pathol 113:104366. https://doi.org/10.1016/j.yexmp.2019.104366 99. Irwandi RA, Khonsuphap P, Limlawan P, Vacharaksa A (2018) Mir-302a-3p regulates rankl expression in human mandibular osteoblast-like cells. J Cell Biochem 119(6):4372–4381. https://doi.org/10.1002/jcb.26456 100. Zhao H, Zhang J, Shao H, Liu J, Jin M, Chen J, Huang Y (2017) Mirna-340 inhibits osteoclast differentiation via repression of mitf. Biosci Rep 37(4):BSR20170302. https://doi.org/10. 1042/BSR20170302 101. He Y, Rhodes SD, Chen S, Wu X, Yuan J, Yang X, Jiang L, Li X, Takahashi N, Xu M, Mohammad KS, Guise TA, Yang FC (2012) C-fms signaling mediates neurofibromatosis type-1 osteoclast gain-in-functions. PLoS One 7(11):e46900. https://doi.org/10.1371/journal. pone.0046900 102. Zhao C, Sun W, Zhang P, Ling S, Li Y, Zhao D, Peng J, Wang A, Li Q, Song J, Wang C, Xu X, Xu Z, Zhong G, Han B, Chang YZ, Li Y (2015) Mir-214 promotes osteoclastogenesis by targeting pten/pi3k/akt pathway. RNA Biol 12(3):343–353. https://doi.org/10.1080/ 15476286.2015.1017205 103. Lou Z, Peng Z, Wang B, Li X, Li X, Zhang X (2019) Mir-142-5p promotes the osteoclast differentiation of bone marrow-derived macrophages via pten/pi3k/akt/foxo1 pathway. J Bone Miner Metab 37(5):815–824. https://doi.org/10.1007/s00774-019-00997-y

Chapter 2

MicroRNAs and the Diagnosis of Osteoporosis Ying Huai, Hui Li, Ye Tian, Airong Qian, and Zhihao Chen

Contents 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 General Overview of miRNAs as Biomarkers for Different Types of Osteoporosis . . . . . 2.2.1 miRNAs as Diagnostic Biomarkers in Primary Osteoporosis . . . . . . . . . . . . . . . . . . . . . 2.2.2 miRNAs as Diagnostic Biomarkers in Secondary Osteoporosis . . . . . . . . . . . . . . . . . . 2.3 Application of miRNAs in the Diagnosis of Osteoporosis and Osteoporotic Fracture Risk Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Advantages and Disadvantages of miRNAs as Novel Biomarker Compared to the Conventional Methods for Osteoporosis Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Clinical Status of miRNAs as Diagnostic Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29 30 30 34 36 38 39 41 41

Abstract Osteoporosis poses an immense burden to the society in terms of morbidity, mortality, and financial cost. Accurately and early diagnosis of the onset of osteoporosis and fracture risk is the key to reduce this burden and deliver effective therapy. Current screening and monitoring approaches for osteoporosis mainly include computed tomography (CT), magnetic resonance imaging, ultra-sound measurements, dual-energy X-ray absorptiometry (DXA), fracture risk assessment tool (FRAX) as well as some bone turnover markers. However, these conventional diagnostics are either limited in specificity and sensitivity, or high-cost. Recently, microRNAs (miRNAs) have been recognized as important regulators of bone physiology and potential diagnostic biomarkers for different types of osteoporosis

Y. Huai · Y. Tian · A. Qian · Z. Chen (*) Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, School of Life Sciences, Northwestern Polytechnical University, Xi’an, China e-mail: [email protected]; [email protected]; [email protected]; [email protected] H. Li Department of Adult Joint Reconstruction, Xi’an Hong-hui Hospital, Xi’an, China © Springer Nature Singapore Pte Ltd. 2021 A. Qian, Y. Tian (eds.), Noncoding RNAs and Bone, https://doi.org/10.1007/978-981-16-2402-5_2

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as well as fracture risk assessment. In this chapter, we will review the potential of miRNAs as diagnostic biomarkers in different types of osteoporosis and osteoporotic fracture. After comparing with the conventional diagnostics, the advantage and disadvantage of miRNAs as indicators in osteoporosis diagnosis will also be discussed. The emerging perspectives of miRNAs as biomarkers maybe a novel strategy for the clinical diagnosis of osteoporosis, which will also provide a significant impact on the early prevention of complex diseases in future. Keywords miRNAs · Osteoporosis · Diagnosis · Biomarkers · Osteoporotic fracture risk assessment

Abbreviations ALP ATF4 AUC BMD BMP2 CT DEXA DOP DXA ELK1 FRAX GCs GIOP HDT HMGA2 MAP9 miRNA MSCs OCN OSX POMP PTH RANKL RICTOR ROC TGFBR1 TRAP VEGFA WHO

Alkaline phosphatase Activating transcription factor 4 Area under the curve Bone mineral density Bone morphogenetic protein 2 Computed tomography Dual-energy X-ray absorptiometry Disuse osteoporosis Dual-energy X-ray absorptiometry ETS domain-containing protein 1 Fracture risk assessment tool Glucocorticoids GC-induced osteoporosis Head-down tilt High mobility group AT-hook 2 Microtubule-associated protein 9 MicroRNA Mesenchymal stem cells Osteocalcin Osterix Postmenopausal osteoporosis Parathyroid hormone Receptor activator of nuclear factor-κB ligand RPTOR independent companion of MTOR complex 2 Receiver operating characteristic curve Transforming growth factor beta receptor 1 Tartrate resistant acid phophatase Vascular endothelial growth factor A World health organization

2 MicroRNAs and the Diagnosis of Osteoporosis

2.1

29

Introduction

Osteoporosis is an increasingly prevailing subclinical degenerative bone disease, characterized by low bone mineral density (BMD), progressive bone loss, and destroyed bone microstructure [1]. Typical signs and symptoms of osteoporosis mainly include pain, stiffness, skeletal hypofunction, and limitation of movement, even companied by an increased risk of bone fractures. With a growing number of patients, osteoporosis has imposed immense burdens to the individuals and society in terms of morbidity, mortality, and financial cost [2, 3]. According to the diagnostic criterion of the World Health Organization (WHO), nearly 200 million people are hampered by osteoporosis annually worldwide, most of which are postmenopausal women and the aged [4]. In China, more than 69.4 million people >50 years were suffered to osteoporosis and approximately 687,000 populations develop to hip fractures each year [5]. Moreover, it has been estimated that the financial costs associated with bone fractures will reach $25.3 billion by the end of 2025 in the USA [6]. Thus, early diagnosis of onset is the key to the effective prevention and therapy for osteoporosis. If increased risk of bone loss is diagnosed prior to the first occurrence of osteoporosis, incidence can be significantly reduced by preventive pharmacologic treatments and/or lifestyle interventions. Traditionally, the commended diagnostic tools for osteoporosis management, including computed to tomography (CT), magnetic resonance imaging, ultra-sound measurements and dual-energy X-ray absorptiometry (DXA) as well as fracture risk assessment tools like FRAX®, constituted the standard for osteoporosis and osteoporotic fracture risk monitoring [7]. Nevertheless, these conventional tools were either expensive or inaccurate. In addition, although some bone metabolism biochemical indicators, such as alkaline phosphatase (ALP), osteocalcin (OCN), and parathyroid hormone (PTH), are also applied in the early diagnosis of osteoporosis, they are unstable inherently and subject to several sources of variability [8]. The mentioned problems of these existing osteoporosis diagnosis methods underscored the utmost importance for the identification of more specific and reliable biomarkers in the early diagnosis of osteoporosis. Fortunately, a novelty kind of RNA, microRNAs (miRNAs) have garnered increased interest and attention from researchers and clinicians for their regulatory roles in gene expression and the homeostasis of complex biological phenotypes [9]. It has become possible to assess the onset and progression of disease through monitoring the changes in miRNA expression based on liquid biopsies, preferably serum or plasma. In terms of osteoporosis, miRNAs are by now well-established regulators of bone remodeling. For example, miR-218 was revealed to exert as a negative regulator in osteoclastogenesis and bone resorption by suppressing the p38MAPK-c-Fos-nuclear factor of activated T-cells (NFATc1) pathway [10]. On the other hand, miR-214 was shown to inhibit osteoblast activity and suppress bone formation by directly targeting activating transcription factor 4 (ATF4) [11]. All these studies hinted at the potential indicative roles of miRNAs in the diagnosis of osteoporosis.

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In this chapter, we will mainly review the potential of miRNAs as diagnostic biomarkers indifferent types of osteoporosis. Compared to the traditional methods, the advantage and disadvantage of miRNAs as indicators in osteoporosis diagnosis will also be discussed.

2.2

General Overview of miRNAs as Biomarkers for Different Types of Osteoporosis

Epidemiological studies have demonstrated that osteoporosis is a complex polygenic disorder with numerous contributing factors, including environmental and genetic risk factors such as hormone secretion, heredity, age, lifestyle, and chronic inflammatory diseases [12]. Generally, based on the various predisposing factors and pathogenetic mechanisms, osteoporosis is categorized into three main types: primary osteoporosis, secondary osteoporosis, and idiopathic osteoporosis. Therein, the primary osteoporosis including senile and postmenopausal osteoporosis is the most common type. Secondary osteoporosis is always induced by other diseases such as gastrointestinal diseases and hematological disease as well as some drug factors like long-term medication of GCs and PTH. The idiopathic osteoporosis, which mainly developed in adolescent population, is rare and related to family medical history or dietary habits. In the last decades, miRNAs as biomarkers were primarily involved in several common osteoporosis, such as hormone relevant osteoporosis, aging-related osteoporosis, and disuse osteoporosis (DOP). In this section, we aimed to unravel the potential of miRNAs as diagnostic biomarkers and summarize how miRNAs exert their roles in several types of osteoporosis. Some representative miRNAs as biomarkers in osteoporosis were listed in Table 2.1.

2.2.1

miRNAs as Diagnostic Biomarkers in Primary Osteoporosis

2.2.1.1

miRNAs and Postmenopausal Osteoporosis

Postmenopausal osteoporosis (POMP) is the most archetypal type of primary osteoporosis affecting up to 40% of postmenopausal women. POMP always poses a serious disorder with significant physical, psychosocial, and financial consequences, which greatly reduce the women’s life quality and arouse increasingly concern by society [30]. POMP is common in women aged >50 years and mainly caused by estrogen deficiency following the menopause. Generally, menopause is accompanied by gradual decline in ovarian estrogen production. Estrogen deficiency removes the inhibition of estrogen on osteoclast activity and contributes to bone loss [31]. The rapid bone loss associated with menopause is impressive. During the 5–7 years surrounding menopause, the bone mass dropped by approximately 12% of

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Table 2.1 Representative miRNAs involved in different types of osteoporosis References Ma et al. [13]

Meng et al. [14] RamírezSalazar et al. [15] Wang et al. [16] Yavropoulou et al. [17] Yavropoulou et al. [17] Yavropoulou et al. [17] Chen et al. [18]

Ma et al. [13]

Chen et al. [19] Ren et al. [20]

Ren et al. [20]

Osteoporosis types POMP

miRNAs miR-4975p miR-181c5p miR-1945p miR-23b3p miR-1403p miR-133a

Expression Down

Function Positively regulated the differentiation and mineralization of osteoblast

Up

Involved in multiple osteoporosis pathways /

POMP

POMP

miR-21-5p miR-29a3p miR-23a3p miR-125b5p miR-30b5pmiR103-3p miR-1423p miR-3283p miR-181c5p miR-497 miR-503

Down

Biomarker and/or regulatory element in circulating monocytes /

POMP

Up

/

POMP

Down

/

POMP

Down

/

POMP

Down

POMP

Hsa-miR214-5p Hsa-miR10b-5p Hsa-miR21-5p Hsa-miR451a Hsa-miR186-5p Hsa-let-7f5p Hsa-let-7a5p Hsa-miR27a-3p

Up

Negative associated with the differentiation and mineralization of osteoblast Negative associated with osteoclastogenesis /

GIOP

Down

/

GIOP

Up

Up

Down

POMP

POMP

(continued)

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Table 2.1 (continued) References Liu et al. [21]

miRNAs miR-106b

Expression Up

Ko et al. [22]

miR-29a

Down

Shi et al. [23]

miR-1792a miR-199a5p miR-216a

Down

Down

miR-125b

Up

miR-4975p miR-181c5p miR-1323p miR-103

Down

Up

miR-33-5p

Down

miR-1393p

Up

Shi et al. [24] Li et al. [25] Chen et al. [26] Ma et al. [13]

Hu et al. [27] Sun et al. [28]

Wang et al. [29] Wang et al. [17]

Up

Up

Function Negatively regulated osteogenic differentiation of mesenchymal stem cells Positively regulate osteogenic differentiation capacity Negatively regulate osteoclastogenesis Increased the inhibition effect of GCs on osteoblast proliferation Positively correlated with bone formation Negatively regulate the proliferation and osteogenic differentiation Positively regulate the differentiation and mineralization of osteoblast

Negatively regulate osteogenic differentiation Inhibit osteoblast proliferation under simulated microgravity condition Positively regulation of osteoblast differentiation Suppress osteoblast differentiation

Osteoporosis types GIOP

GIOP GIOP GIOP GIOP Age-related osteoporosis Age-related osteoporosis

DOP DOP

DOP DOP

postmenopausal women. Thus, there is a need to develop novel sensitive and specific biomarkers for early diagnosis of postmenopausal osteoporosis to improve the health of postmenopausal women. In recent decades, evidences have shown that miRNAs mediate the functions of bone cells such as chondrocytes, osteoblasts, and osteoclasts, as well as bone metabolism. Numerous miRNAs have been revealed to participate in bone remodeling and the pathological process of postmenopausal osteoporosis. For example, our recent study showed that miR-181c-5p and miR-497-5p were downregulated in the serum of postmenopausal women with osteopenia or osteoporosis, suggesting that circulating miR-181c-5p and miR-497-5p might act as potential biomarkers for monitoring effects of anti-osteoporosis therapies or diagnostic approach [13]. Wang et al. reported that miR-133a was over-expressed in patients with postmenopausal osteoporosis and involved in the regulation of postmenopausal osteoporosis through promoting osteoclast differentiations, such that miR-133a could be considered as a potential biomarker for the diagnosis of osteoporosis [16]. Another miRNA associated with postmenopausal osteoporosis was miR-152, which was up-regulated during postmenopausal osteoporosis and restrained the

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osteoblast differentiation by inhibiting the expression of RPTOR Independent Companion of MTOR Complex 2 (RICTOR) [32]. Besides, Ramírez-Salazar and coworkers identified miR-140-3p and miR-23b-3p as potential biomarkers for osteoporosis and osteoporotic fracture in postmenopausal women [15]. Researchers observed a significant enrichment of circulating miR-338 cluster in postmenopausal osteoporosis patients, indicating that miR-338 cluster in serum could serve as a promising diagnostic and therapeutic target for postmenopausal osteoporosis patients [33]. Moreover, a research performed by Kong et al. showed that miR-320a was highly expressed in postmenopausal osteoporosis and negatively regulated osteoblasts differentiation by reducing microtubule-associated protein 9 (MAP9) and inhibiting PI3K/AKT signaling pathway [34]. Another up-regulated miRNA in the low BMD group —miR-422a in human circulating monocytes (osteoclast precursors) was a potential biomarker for postmenopausal osteoporosis [35]. In addition, enhanced miR-194-5p expression in women with osteoporosis was detected and identified as a viable biomarker for postmenopausal osteoporosis [14]. A recent finding showed that the over-expressed miR-16-5p involved in the regulation of postmenopausal osteoporosis by directly targeting vascular endothelial growth factor A (VEGFA) [36]. In brief, numerous miRNAs are involved in the regulation and management of postmenopausal osteoporosis, displaying their potential as biomarkers for the early detection of postmenopausal osteoporosis.

2.2.1.2

miRNAs and Senile Osteoporosis

Aging is always accompanied with the defects in the coupling of bone resorption and bone formation, causing deteriorated bone microstructure and serious bone loss [37]. Specifically, senescence of cells in the bone leads to loss of the bone and osteoporosis in aged people [38]. With accelerated aging, osteoblasts exhibited impaired cell activity and reduced differentiation capacity as well as mineralization capacity. Besides, there are positive age-dependent changes in osteoclasts activity and the expression of osteoclast differentiation factors. Osteocytes also showed degenerative changes with decreased osteocyte cell population and lacunar density during aging. Age-related osteoporosis, also called senile osteoporosis, has become a worldwide bone disease with the aging of the world population. Generally, bone mass loss due to aging increases the risk of osteoporosis and osteoporotic fracture [39]. Fractures in aged people are responsible for impaired quality of life, as well as significant morbidity, mortality, and loss of independence. Therefore, the early diagnosis and prevention is of essential for age-related osteoporosis. Recently, a number of miRNAs have already been identified to play a role in the diagnosis of age-related osteoporosis. There is evidence that miR-141 was differentially expressed in mesenchymal stem cells (MSCs) with aging and senescence, which further regulate the process of MSCs differentiation into osteoblasts [40]. A study performed by Li et al. has shown that miR-188 was a key regulator of the age-related switch between osteogenesis and adipogenesis of BMSCs and may represent a potential diagnostic biomarker and therapeutic target for age-related bone loss

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[41]. Additionally, Xu et al. found that the expression of miR-31a-5p was markedly higher in BMSCs-derived exosomes from aged rats compared to those from young rats, which decreased the osteoblastic activity and promoted the osteoclastic activity, thus resulting in age-related osteoporosis [42]. Li and coworkers revealed that the elevated osteoclast-derived exosomal miR-214-3p expression inhibits osteoblastic bone formation in elderly women with fractures [43]. We concluded that several miRNAs have been identified as regulators and promising biomarkers in the development of senile osteoporosis.

2.2.2

miRNAs as Diagnostic Biomarkers in Secondary Osteoporosis

2.2.2.1

miRNAs and Drug-Induced Osteoporosis

Among the multiple pathogenic factors of secondary osteoporosis, PTH and GCs were the most common and closely related to the pathogenesis of osteoporosis. Intermittent administration of PTH, a natural hormone, could stimulate bone formation and anabolism. Nevertheless, long-term administration of PTH maintains the serum calcium levels by promoting the osteoclast differentiation and bone resorption, thus leading to bone loss [44]. Excessive administration of GC could inhibit the osteoblastic differentiation and bone formation, as well as increase the number of osteoclasts and bone-resorbing sites, thus inducing severe bone loss, named GC-induced osteoporosis [45]. In this section, the potential of miRNAs as early diagnostic biomarker in the secondary osteoporosis is reviewed. GCs are extensively applied for the treatment of immune and inflammatory disorders due to their powerful immune suppressive and anti-inflammatory actions. However, long-term exogenous GC therapy might cause various adverse effects; especially rapid and pronounced bone loss and subsequently GC-induced osteoporosis (GIOP) [46]. The pathogenesis of this type osteoporosis was the reduction of bone formation and the increase of bone resorption induced by GCs [47]. GIOP is the most frequent secondary cause of osteoporosis and the third most common etiology of bone loss in postmenopausal women and aging individuals [48]. Emerging evidence has demonstrated that bone fragility and fractures induced by GIOP is much more severe than that induced by postmenopausal osteoporosis, and GIOP has higher disability and mortality rates [49]. Thus, it is of quite importance and urgency to explore accurate and specific biomarkers for the diagnosis and treatment of GIOP. Recently, the dysregulation of miRNAs in GIOP has attracted broad attention of researchers and was considered as the potential biomarkers for GIOP. For example, over-expressed miR-106b was found to inhibit osteoblastic differentiation and bone formation partly through directly targeting bone morphogenetic protein 2 (BMP2) in the GIOP, although it was limited to the animal or cellular level and there could be some species differences in miRNA expression [50]. Another study identified nine differentially expressed miRNAs in GIOP patients, including six up-regulated

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miRNAs (hsa-miR-186-5p, hsa-miR-21-5p, hsa-mir-214-5p, hsa-miR-10b-5p, hsa-miR-451a, and hsa-mir-novel-chr3_49413) and three down-regulated (hsa-let7f-5p, hsa-let-7a-5p, and hsa-mir-27a-3p) [51]. Additionally, the expression of miRNAs, such as miR-29a, miR-34a-5p, and miR-199a-5p, was also modulated by the excessive glucocorticoids, which further regulated the proliferation and differentiation of osteoblast lineage cells [20]. Zhang et al. detected that the GC-associated miR-338-3p was down-regulated in the GIOP model and aggravated GC-induced osteoclast formation and bone resorption by targeting receptor activator of nuclear factor-κB ligand (RANKL) [52]. Recently, Shen and coworkers revealed that let-7f-5p was down-regulated in the vertebrae of patients with GIOP and involved in GC-inhibited osteoblast differentiation through increasing the expression of its target transforming growth factor beta receptor 1 (TGFBR1) [53]. In brief, there are increasingly dysregulated miRNAs were identified to be associated with the pathogenesis of GIOP and served as the marker for GIOP diagnosis and therapy.

2.2.2.2

miRNAs and Disuse Osteoporosis

There are many lines of evidence indicating that the stimulus of mechanical stress is indispensable for maintaining the positive balance of bone metabolism and bone growth [54]. Appropriate mechanical stimulus for the skeleton stimulated bone remodeling and accelerated fracture healing through up-regulating the osteogenic factors such as ALP, Runx2, and osterix (Osx), but inhibiting the expression of RANKL, tartrate resistant acid phosphatase (TRAP), and other bone resorption factors. However, the lack of mechanical stimulus, as experienced by bedridden patients or astronauts in microgravity conditions, results in profound bone metabolism disorder and bone loss, leading to osteoporosis, called disuse osteoporosis [55]. It was known that mechanical unloading or microgravity stimulated abnormal activation of osteoclasts and restrained the activity of osteoblasts, leading to bone tissue destruction and thus disuse osteoporosis [56]. Nevertheless, disuse osteoporosis was often masked by other obvious clinical symptoms, which may further develop to an osteoporotic fracture. Therefore, it is a pressing need to explore more convenient and accurate diagnostic biomarkers for better early detection and treatment of disuse osteoporosis. The present section reviewed the expression of miRNAs and their mechanism of action in mechanical unloading regulated bone metabolism to further explore their potential as diagnostic markers in disuse osteoporosis. Evidences are increasing that the underlying regulatory mechanism for disuse osteoporosis may be related to miRNAs. Mechanical unloading was reported to induce significant expression changes of miRNAs that modulate the expression of osteogenic and bone resorption factors, which suggested that miRNAs might play an important role in mechanical unloading mediated bone metabolism [57]. A series of miRNAs have been identified to be sensitive to mechanical unloading and have a marked effect on bone metabolism. Sun and coworkers observed that the up-regulated expression of miR-103 induced by simulated microgravity could repress Calcium Voltage 1.2 (Cav1.2)

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expression, resulting in the inhibition of osteoblasts proliferation [58]. Besides, it is reported that the expression of miR-33-5p was inhibited by microgravity, thus restraining osteoblast differentiation by targeting high mobility group AT-hook 2 (HMGA2) [28]. Moreover, the overexpression of miRNA-132-3p induced by stimulated microgravity was found to suppress osteoblast differentiation by targeting the gene encoding E1A-binding protein p300, a histone acetyltransferase important for the activity and stability of Runx2 [29]. Notably, a recent research showed that miR-139-3p was up-regulated in the femurs of hindlimb unloading mice and MC3T3-E1 cells under simulated microgravity, thus further suppressing osteoblast differentiation and promote osteoblast apoptosis by targeting ETS domaincontaining protein 1 (ELK1) and interacting with long noncoding RNA ODSM [27]. Therefore, these findings above suggested that miRNAs might be directly linked to bone tissue homeostasis in the lack of mechanical stimulus and serve as a novel biomarker of bone turnover in osteoporotic patients, which indicating the potential value of miRNAs in disuse osteoporosis diagnosis.

2.3

Application of miRNAs in the Diagnosis of Osteoporosis and Osteoporotic Fracture Risk Assessment

Over the past decade, miRNAs as novel diagnostic biomarkers for many pathological processes such as cancer, Alzheimer’s, sepsis, and rheumatoid arthritis has aroused extensive concerns [59–62]. Generally, miRNAs that were bound to proteins or encapsulated in exosomes and/or micro vesicles are released into the bloodstream and biological fluids where they could be detected by highly sensitive and specific methods (such as quantitative PCR or next-generation sequencing). Moreover, as highly sensitive fine-turners of biological processes, miRNAs could reliably prognosticate a subject-specific risk of disease onset, progression and response to therapy. As such, miRNAs provide as a prompt and easily accessible biomarker to determine the subject-specific epigenetic environment of a specific condition and open new frontiers in personalized medicine. Recently, the potential roles of miRNAs in skeletal system and related diseases were gradually as the emerging hot topic for bone biologists and orthopedists. While the multiple roles of miRNAs in bone pathophysiology have been identified and validated, their roles in the clinical diagnosis of bone diseases have not yet been established. This section aimed to describe the potential use of bone-associated circulating miRNAs as biomarkers for determining predisposition, onset, and development of osteoporosis and the related risk of osteoporotic fracture. For the past few years, numerous miRNAs have been identified as the diagnostic markers of osteoporosis with highly sensitivity and specificity in the detection of different types of osteoporosis. A recent study of our group found that, the AUCs of

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miR-181c-5p and miR-497-5p in identifying osteoporotic subjects from normal healthy subjects were 0.87 (P < 0.001) and 0.92 (P < 0.001), respectively, suggesting that miR-181c-5p and miR-497-5p might exert as biomarkers with satisfied diagnostic power in discriminating osteopenia and osteoporosis patients from normal [13]. Besides, Chen et al. found that the serum levels of four miRNAs (miR-30b-5p, miR-103-3p, miR-142-3p, and miR-328-3p) in osteoporosis patients were positively correlated with BMD. And the ROC analysis proved their diagnostic potential for osteoporosis based on their AUC values (all >0.75) [18]. Another study conducted by Ramírez-Salazar pointed the higher serum levels of miR-140-3p and miR-23b-3p in the osteopenia or osteoporosis groups, suggesting they might be as potential biomarkers candidates for osteoporosis [15]. Moreover, Seelinger et al. revealed that miR-100, miR-125b, miR-21, miR-23a, and miR-24 were up-regulated both in serum and bone tissues and showed significant AUC, sensitivity, and specificity in discriminating osteoporosis from non-osteoporosis subjects, suggesting their potential as biomarkers for osteoporosis and related hip fractures [63]. A case-control study performed by Kocijan identified 8 differentially expressed serum miRNAs (miR-140-5p, miR-152-3p, miR-19a-3p, miR-19b-3p, miR-30e-5p, miR-324-3p, miR-335-5p, and miR-550a-3p) in postmenopausal osteoporosis, which exerted a high discriminating power between individuals with bone fracture and healthy individuals (AUC > 0.9). Notably, based on the identified miRNAs in osteoporosis patients, Kocijan et al. have developed a laboratory assay of miRNAs test, which provides reagents and software to quantify miRNAs in human serum and to calculate a score for fracture risk assessment based on the relative abundances of the included miRNAs [64]. Another research discovered that the up-regulation of miR-122-5p, miR-125b-5p, and miR-21-5p consistently discriminated between the osteoporosis patients with fractures and controls, and ROC analysis confirmed their diagnostic potential in postmenopausal osteoporosis (AUC 0.87 for miR-122-5p, 0.76 for miR-125-5p, and 0.87 for miR-21-5p) in accordance with previous observations [65]. As reviewed above, the growing body of evidence revealed the fundamental regulatory roles exerted by miRNAs in bone homeostasis, along with aberrant expression in osteoporosis onset, highlighting their huge potential as diagnostic biomarkers in osteoporosis and fracture risk assessment. Unfortunately, clinical studies on the identification of miRNAs as diagnostic biomarkers for osteoporosis have adopted various different experimental protocols, making the obtained results were diverse from different cases or labs. Furthermore, the great majority of the published clinical studies are featured by limited sample sizes. Another limitation is that the analyses of some differentially expressed miRNAs in osteoporosis were limited to the animal or cellular level, and no clinical study was conducted. Thus, more attention should be posed to the standardization of miRNAs discovery and validation to obtain valuable biomarkers for clinical practice. The diagram of miRNAs as biomarkers in the diagnosis of osteoporosis is shown in Fig. 2.1.

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Fig. 2.1 The diagram of miRNAs as biomarkers in the diagnosis of osteoporosis

2.4

Advantages and Disadvantages of miRNAs as Novel Biomarker Compared to the Conventional Methods for Osteoporosis Diagnosis

The measurability criterion and the used apparatus for diseases require an accurate and reproducible analytical method that could provide reliable measures rapidly and at reasonable cost. Generally, biochemical markers of bone turnover for osteoporosis provide a means of evaluating skeletal dynamics that complements static measurements of BMD by DXA, CT or ultra-sound measurements [66]. Unfortunately, conventional markers have some limitations including a lack of specificity for bone tissue, their inability to reflect osteocyte activity or periostea lap position. In addition, they do not allow the investigation of bone tissue quality, an important determinant of skeletal fragility. Moreover, traditional clinical measurement methods of BMD such as CT, magnetic resonance imaging, and ultra-sound measurements are not accurate or sensitive. Although DEXA is highly accurate, it involves complex equipment, which is bulky, expensive, and emits radiation [7]. It also should be mentioned that FRAX tools usually considered other clinical risk factors apart from BMD, thus existing individual variation. Besides, some of the FRAX®-specific risk factors are partially or wholly independent of BMD, providing information on fracture risk above that of BMD alone [67]. The introduction of novel clinical measurement indicators, miRNAs, is therefore in the interest of the public health and a willingness of the health care payers. Recently, the use of circulating (or also tissue) miRNAs as biomarker is nearly ready for implementation in clinical practice and has aroused broad attentions. Notably, miRNAs also show advantages as diagnostic biomarkers for osteoporosis when compared with the traditional methods [7]. As epigenetic regulators of gene expression, miRNAs act as modulators rather than effectors of a specific biological function, and they provide a prompt and easily accessible tool to determine the

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epigenetic environment of a specific condition, thus being considered a personalized signature for disease diagnosis. In comparison with FRAX® tool and DXA, the novel molecular diagnostic method miRNAs test is both highly sensitivity of screening and monitoring bone fracture risk and cost-effective. Nevertheless, the miRNAs test is not intended to completely replace FRAX® tool and DXA, owing that they have been widely used and safely established in clinical practice as well as provide the criterion required for osteoporosis diagnosis according to the WHO. Conversely, the miRNAs test is only aimed to complement the BMD measurement, which is not a suitable and sufficiently accurate method for identifying high-risk osteoporosis patients, and offer orthopedists clinical references for osteoporosis diagnosis and treatment decisions. Another superiority of miRNAs is that they are easily detectable and stability in biological fluids such as plasma, serum and urine, which are sources of biomarkers with broad applicability in clinical research. These advantages notwithstanding, there are still some weakness in the use of circulating (or also tissue) miRNAs as biomarkers. Specifically, miRNAs evaluation was affected by several variables in different analysis phases. In the pre-analytical phase, there are two sets of factors that affect miRNAs evaluation: patient-related (lifestyle habits and diseases) and sampling-related (source/matrix, sample collection, and handling) factors. Smoking, physical activity, diet, head-down tilt (HDT) bed rest and circadian rhythm could alter the level of a specific miRNA in circulation [68–72]. Moreover, during sample collection and handling, miRNA quantification may be affected by the type of collection tube and anticoagulant coating, in addition to blood cell count, needle gauge [73], and hemolysis. As for the analytical phase, the analytical protocols and platforms choice will affect the reproducibility and specificity for the discovery and the validation of a biomarker. The major postanalytical influences for miRNAs evaluation are data normalization and choice of the right reference gene. For these problems, specific guidelines to standardize pre-analytical, analytical, and post-analytical variables are desirable to obtain reliable and comparable miRNA expression data and to accelerate the definitive clinical implementation of miRNAs-based tests (Table 2.2).

2.5

Clinical Status of miRNAs as Diagnostic Markers

Even with many advantages of miRNAs as biomarkers for various diseases, the actual application of miRNA in clinical diagnosis remains at the stage in laboratory. At present, how to apply them to clinical practice becomes a major dilemma. Recently, numerous researchers are concentrating on this work. It is reported that a DNA probe (Probe) capable of specifically recognizing the target miRNA was designed to detect the electrical signal and observe the stability of the signal through the nanochannels. This new method has been for early detection of pancreatic cancer-related microRNAs and is expected to be successfully applied in clinical practice in the near further [74]. Analogously, Xu et al. also showed that they have designed a novel DNA circle capture probe with multiple target recognition domains

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Table 2.2 Comparison of miRNAs and the conventional diagnostic tools Tools miRNAs [7]

Conventional diagnostic tools

Dual-energy X-ray absorptiometry (DEXA) [7] Computed tomography (CT) [66] Magnetic resonance imaging [66] Ultra-sound measurements [66] FRAX® tool [67]

Bone turnover markers [66]

Advantages High accuracy Prompt and easily accessible Cost-effective High accuracy

Cost-effective Cost-effective Cost-effective Cost-effective

High operability Cost-effective

Disadvantages Susceptible to variables in different analysis phase

Undetectable in real time costly Operationally complex Not accurate or sensitive undetectable in real time Not accurate or sensitive undetectable in real time Not accurate or sensitive undetectable in real time Undetectable in real time Individual variation Inaccurate Unstable and inaccurate

of the miRNAs to develop an electrochemical biosensor for ultrasensitive detection of miRNAs in cancer cell lysates. The present strategy paved a new path in the applications of miRNAs as biomarkers in clinical diagnostic of diseases [75]. Another study pointed that integrating exosomal microRNAs and electronic health data could potentially improve clinical diagnosis of tuberculosis, which might also be applied to the diagnosis of other diseases [76]. These researches hinted that miRNAs as biological signals could be converted to electrical signals to be detected in clinical application. In addition, the constantly emerging of new platforms for the detection of miRNA expression profiles has facilitated the application of miRNA in clinical diagnosis. For example, in the past several years, Hwu et al., introduced a new-type analysis tool based on dark-field microwells to detect the direct miRNA sensing with gold nanoparticles. They have demonstrated the feasibility of the darkfield microwells to detect miRNA in both buffer solution and cell lysate, which implied its promising for clinical diagnosis [77]. In addition, Konno and coworkers pointed that the distinct methylation levels of mature miRNAs might be a promising diagnostic strategy in cancer [78]. Specifically, a novel MALDI-TOF-MS tool was used to detect the methylated bases of miRNAs through conventional mass spectrometry to capture cancerization. If these novel tools are proved to be highly effective in clinical tests, they might be applied to the diagnosis for other diseases, such as osteoporosis. Thus, in my view, combining miRNAs with the new-type tools, other signals or special materials may be the promising prospect of miRNAs as diagnostic biomarkers in clinical application.

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41

Conclusion

MiRNAs as a class of evolutionarily conserved endogenous noncoding RNAs, play important roles in diverse physiological and pathological processes, including cell differentiation, proliferation, apoptosis, and cancer development. Given the crucial roles that miRNAs appear to exert in bone metabolism and bone homeostasis, exploring their potential has recently garnered increased interest. Plenty of differentially expressed miRNAs as reviewed above have been identified in bone samples from osteoporosis patients, which has led to progress in developing miRNAs as diagnostic tools and might provide clinical signals for understanding the complex mechanisms of osteoporosis. Moreover, combining miRNAs with the new-type tools, other signals or special materials may be the promising prospect of miRNAs as diagnostic biomarkers in clinical application. However, more clinical investigations are required to further address possible issues regarding the application of miRNAs as biomarkers. Acknowledgements All the authors of this chapter are immensely grateful to their respective universities and institutes for their technical assistance and valuable support in the completion of this manuscript. This work was supported by the Natural Science Foundation of China (82072106, 31570940, 31370845, 81772017, and 31400725), the China Postdoctoral Science Foundation (2020 M683573, 2018 T111099, 2017 M610653 and 2017 M613210), the New Century Excellent Talents in University (NCET-12-0469), the Project Supported by Natural Science Basic Research Plan in Shaanxi Province of China (2020JM-100, 2018JM3040 and 2018SF-263), the Innovation Foundation for Doctor Dissertation of Northwestern Polytechnical University (CX201821).

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Chapter 3

MicroRNAs and Osteoarthritis Shenxing Tan, Qian Huang, Xuechao Liang, Airong Qian, and Ye Tian

Contents 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 MicroRNAs and Chondrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 miRNAs and the Degenerative Changes of Extracellular Matrix of Articular Cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 miRNAs and Chondrocyte Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 miRNAs and Joint Pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 microRNA Regulatory Pathways in OA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.1 NF-κB Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.2 TGF-β Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.3 Wnt Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.4 PI3K/Akt Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.5 p53 Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Early Diagnosis and Treatment Using microRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.1 miRNAs and Osteoarthritis Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.2 miRNAs and Osteoarthritis Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

50 55 58 59 60 61 62 62 63 63 64 65 65 66 67 68

Abstract Osteoarthritis (OA) is the most common joint disease. It is characterized by degenerative changes of articular cartilage and chondrocyte apoptosis. The prevalence of OA increases with age and affects most individuals over the age of 65. However, due to the various influencing factors and complicated molecular mechanisms of OA pathogenesis, at present, there is no effective method to cure OA except total joint replacement surgery. Interventions could be taken to slow the progression of OA and prevent the irreversible degradation of cartilage. MicroRNAs

S. Tan · Q. Huang · X. Liang · A. Qian · Y. Tian (*) Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China e-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2021 A. Qian, Y. Tian (eds.), Noncoding RNAs and Bone, https://doi.org/10.1007/978-981-16-2402-5_3

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(miRNAs) are endogenous noncoding single-stranded small molecule RNAs, which involve in a series of important processes in life. In recent years, more and more researchers have found that many miRNAs are inseparable from the occurrence of OA. This chapter mainly focuses on summarizing those microRNAs which participate in the occurrence and development of OA (e.g., the proliferation and apoptosis of chondrocytes, the degenerative changes of articular cartilage extracellular matrix (ECM), the process of chondrocyte inflammation and joint pain) as well as the related signal regulation pathways. We will discuss the latest research progress of microRNA in the treatment of OA and provide new insights into potential molecular targets for the prevention and treatment of OA. Keywords microRNAs · Osteoarthritis · Pathways · Chondrocytes · ECM · Chondrocyte inflammation · Joint pain

Abbreviations ACLT ADAMTS AKT ATG-10 ATG2B AUCs Bcl-2 CCL4 COL2A1 COMP COX-2 CTGF CTX-2 CX43 DNMT1 DVL3 ECM EIF4G2 ERG FRAT2 FUT4 FZD3 FZD5 GDF-5 hADSCs HCs HDAC2 HDAC4 HIF-1α

Anterior cruciate ligament transection A disintegrin and metalloproteinase with thrombospondin motifs Protein kinase B Autophagy related 10 Autophagy related 2b Area under the curve Bcl-2 isoform X2 Chemokine ligand 4 Collagen type II alpha 1 chain Cartilage oligomeric matrix protein Cyclooxygenase 2 Connective tissue growth factor Type 2 collage Connexin 43 DNA methyltransferase 1 Dishevelled segment polarity protein 3 Extracellular matrix Eukaryotic translation initiation factor 4 gamma 2 Ets transcription factor erg Frat regulator of Wnt signaling pathway 2 Fucosyltransferase 4 Frizzled class receptor 3 Frizzled class receptor 5 Growth differentiation factor 5 Human adipose stem cells Healthy controls Histone deacetylase 2 Histone deacetylase 4 Hypoxia inducible factor 1 subunit alpha

3 MicroRNAs and Osteoarthritis

HIF-2α HMGB1 hUC-MSCs IGF IL-1β IL-6 INK4a iNOS KO KOA LDL Lef-1 LPS MALAT1 MAPK Matn3 MCT miRNA MMP MMP-13 MRI NF-κB NG-R1 NHAC-kn NRF1 OA OSM p38/Mapk p65 PAK2 PBMCs PEC PGE2 PGRN PI3K PIK3R1 PIK3R12 PPARα Runx2 SHIP-1 SIRT1 SMAD2 SMAD3 SOX6 SOX9 SPHK1

Hypoxia inducible factor 2 subunit alpha High mobility group box 1 Human umbilical cord mesenchymal stem cells Insulin like growth facto Interleukin 1 beta Interleukin 6 Cyclin dependent kinase inhibitor 4a Inducible nitric oxide synthase Knockout Knee osteoarthritis Low-density lipoprotein Lymphoid enhancer binding factor 1 Lipopolysaccharide Metastasis associated lung adenocarcinoma transcript 1 Mitogen-activated kinase-like protein Matrix protein matrilin 3 Murine chondrocytes MicroRNA Matrix metalloproteinase Matrix metallopeptidase 13 Magnetic resonance imaging Nuclear factor kappa-B Notoginsenoside R1 Normal human articular cartilage-knee Nuclear respiratory factor 1 Osteoarthritis Oncostatin M Mitogen-activated protein kinase Rela proto-oncogene, Nf-Kb subunit P21-Activated kinase 2 Peripheral mononuclear blood cells The primary extraosseous chondrocytes Prostaglandin E2 Progranulin Phosphatidylinositol 3-kinase Phosphoinositide-3-kinase regulatory subunit 1 Phosphoinositide-3-kinase regulatory subunit 2 Peroxisome proliferator activated receptor alpha Runt related transcription factor 2 Inositol polyphosphate-5-phosphatase D Sirtuin 1 Smad family member 2 Smad family member 3 Sry-box transcription factor 6 Sry-box transcription factor 9 Sphingosine kinase-1

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SPP1 TCF-4 TGF-β Timp2 TLR7 TNF-α WNT5B

3.1

Phosphoprotein 1 Transcription factor 4 The transforming growth factor-Β Metalloproteinase 2 Toll like receptor 7 Tumor necrosis factor alpha Wingless-type MMTV integration site family member 5B

Introduction

The joint is a form of indirect connection between bones. In normal life, due to the coordinated operation of various joints, people can move flexibly. The joint tissue is highly cellular and isotropic at birth, as the tissue matures, the special areas will form. This special structure allows the articular cartilage to withstand significant shear and compressive forces throughout a joint’s range of motion [1, 2]. OA is defined as the syndrome of joint and dysfunction caused by substantial joint degeneration [3]. Characteristics of OA typically include breakdown of the articular cartilage, synovitis, chondrocyte apoptosis, inflammation, and remodeling of the subchondral bone [4]. In general, OA is the most common joint disease and is one of the most frequent and symptomatic health problems for middle-aged and older people [5]. OA is a major source of pain, disability, and socioeconomic cost worldwide [6]. With genetic, biological, and biomechanical components, the epidemiology of OA is complicated and multifactorial. There is still no cure for OA. However, treatment can reduce pain, correct deformities, and improve joint function so as to improve the quality of the patient’s life. Traditionally, a combination of non-drug and drug treatments may be chosen, because it is superior to drug treatment alone. Surgical treatment is appropriate when conservative therapy fails or is inadequate. There are various options for surgical treatment of OA, including extirpation of loose bodies, arthrotomy and joint debridement, osteotomy, joint fusion, and artificial joint replacement [7]. Although functional outcomes can be poor and the lifespan of prostheses is limited, joint replacement is still an ultimate means for symptomatic end-stage disease. Besides, some physiotherapies may be taken to relieve pain and symptoms of OA, e.g., thermotherapy, acupuncture, massage to increase local blood circulation and reduce inflammatory reactions. MicroRNAs (miRNAs) are small single-stranded RNAs, which negatively regulate gene expression [8]. For the biogenesis of miRNAs in vivo, it is described in Chap. 1. More and more researchers have found that many miRNAs participate in occurrence and progression of OA [9]. The recent studies have revealed that RNA interacted with RNA-binding proteins to regulate gene transcription and protein translation and is involved in various pathological processes of OA, thus becoming a potential therapeutic strategy for OA [10] (Table 3.1). MiRNAs mainly participate

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Table 3.1 miRNAs related to osteoarthritis miRNA miR-1 [11]

Target gene HDAC4

miR-7 [12]

IL-1β

miR-9 [13]

IL-6, MMP-13

miR16-5p [14]

SMAD3

miR19b [15]

MALAT1

ATDC5 mouse chondrocyte cells

miR-20 [16]

ATG-10

Human primary chondrocytes (knee)

miR-21 [17]

GDF-5

miR-22 [18] miR23a-3p [19]

PPARα

miR-24 [20]

INK4a

miR26a-5p [21] miR26b [22]

iNOS

Human articular chondrocytes (CH8), human primary chondrocytes (knee) Human primary chondrocytes (joint) Human primary chondrocytes (knee), human chondrosarcoma cells (SW1353) Human primary chondrocytes (knee), human bone marrow mesenchymal stromal cells, one-month-old Ink4a knockdown transgenic mice Human primary chondrocytes (joint)

SMAD

FUT4

Cell/Animal model 17-day-old chick primary chondrocytes (sternum growth plate), murine chondrocytes (MCT), HDAC5 and HDAC9 knockout mice C28/I2 (human normal chondrocytes)

8-week-old male SD rats, human primary chondrocytes (knee) Human primary chondrocytes (knee)

Human primary chondrocytes (knee), SW1353 (human chondrosarcoma cells)

Function miR-1 targets HDAC4 mRNA to inhibit HDAC4 protein levels to promote chondrocyte differentiation Upregulation of miR-7 promotes inflammation and apoptosis of OA chondrocytes miR-9 enhances the proliferation of knee OA chondrocytes miR-16-5p is an important regulator of SMAD3 expression in human chondrocytes Regulation of miR-19 can reduce LPS-induced inflammatory damage of chondrocytes in mice miR-20 inhibits chondrocyte proliferation and autophagy Overexpression of miR-21 may inhibit cartilage formation

Signaling pathway

NF-κB1

Wnt/β catenin, NF-κB PI3K/AKT/ mTOR TGF-β

miR-22 inhibits the growth of chondrocytes miR-23a-3p inhibits the synthesis of chondrocytes and promotes the development of OA Downregulation of miR-24 leads to chondrocyte senescence

IL-lβ

miR-26a-5p regulates chondrocyte proliferation and survival rate miR-26b can inhibit the expression of nuclear mitogen subunit α3

NF-κB

NF-κB

(continued)

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Table 3.1 (continued) miRNA miR27a-3p [23] miR-29 [24]

Target gene MMP-13

Cell/Animal model Human primary chondrocytes (knee)

Function miR-27a-3p is a modulator of ADAMTS5

FZD3, FZD5, DVL3, FRAT2 ERG

C57BL/6 male mice, SW1353 cells, human primary chondrocytes (knee) SW1353 cells

TGF-β1

Human primary chondrocytes (knee)

miR-29 adds an additional level of regulation to the pathway of cartilage homeostasis miR-30b downregulates the expression levels of COL2a and aggrecan miR-33a can promote OA phenotype

SIRT1

SD rats, human primary chondrocytes (knee)

miR92a-3p [28]

HDAC2

miR-93 [29]

OSM

miR-98 [30]

TNFα

Human chondrocyte mesenchymal stem cells (hMSCs), human primary chondrocytes (knee) Eight-week-old C57BL/6 male mice, mice primary chondrocytes (articular cartilage inside the tibia) Female SD rat

miR30b [25] miR33a [26] miR-34 [27]

miR101 [31] miR103 [32] miR105 [33] miR124 [34] miR125b [35] miR126 [36]

SOX6

ADAMTS

ADAMTS4

PIK3R2

SD rat, SD rat primary chondrocytes (femur and tibia) Human primary chondrocytes (cartilage samples for total joint replacement surgery) Human primary chondrocytes (knee) OA mouse model, bone marrow mesenchymal stem cells Human primary chondrocytes (knee) Rabbit cartilage

Signaling pathway NF-κB

SMAD NF-κB, Wnt

TGF-β1/Akt/ SREBP-2

Lentiviral vector against miR-34a sequence can improve OA miR-92a-3p promotes the expression of relative cartilage matrix

SIRT1/p53

miR-93 inhibits cell apoptosis and the production of pro-inflammatory factors miR-98 promotes chondrocyte apoptosis and cartilage degradation Overexpression of miR-101 reduces the level of gene expression miR-103 promotes the development of OA

TLR4 /NF-κB

miR-105 expression is downregulated in OA patients Curcumin may slow down the progression of OA by regulating miR-124 Low expression of miR-125b in OA chondrocytes miR-126 affects the regeneration of rabbit cartilage

IL-1β

SOX9

NF-κB, TLR9

IL-1β

MAPK

(continued)

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Table 3.1 (continued) miRNA miR132 [37] miR137 [38] miR138 [39] miR140 [40] miR142-3p [41] miR145 [42] miR146 [43] miR148a [44]

miR149 [45] miR152 [46] miR155 [47] miR181b [48]

Target gene p65

Cell/Animal model ATDC5 cells

ADAMTS5

Human primary chondrocytes (knee)

p65

Human primary chondrocytes (knee), SW1353 cells

MMP-13

Human cartilage C28/I2 cells

HMGB1

Male C57BL/6 mouse, mouse primary chondrocytes Human primary chondrocytes (femur, tibia and articular cartilage) Primary chondrocytes of SD rats

MMP-13

DNMT1

Human primary chondrocytes (knee)

TNF-α

SW1353 cells

TCF-4

Rat anterior cruciate ligament transected model (ACLT) miR-155 deficient mice

SHIP-1, TNF-α MMP-13

miR199a [49]

COX-2

miR203 [50]

Type II collagen (CTX-II)

Human primary chondrocytes (knee), disproportionate micromelia (DMM) mouse model Human primary chondrocytes (cartilage samples for total joint replacement surgery) OA rat model

Function Upregulation of miR-132 can protect ATDC5 cells from LPS attack miR-137 inhibits cell growth and extracellular matrix degradation The low expression of miR-138 enhances the destruction of OA cartilage miR-140 acts as a negative feedback regulator of MMP-13 in human OA miR-142-3p inhibits chondrocyte apoptosis and inflammation in OA miR-145 regulates the expression of key cartilage genes in human articular chondrocytes miR-146a can promote OA

Signaling pathway NF-κB

NF-κB

IGF, NF-κB

NF-κB

SOX9

IL-1β, TGF-β

Overexpression of miR-148a promotes cartilage production and inhibits cartilage degradation miR-149 is downregulated in OA chondrocytes miR-152 reduces the progression of OA miR-155 is related to cell differentiation and activation of the immune system miR-181b is a negative regulator of chondrocyte differentiation miR-199a is negatively correlated with COX-2 in human chondrocytes

p38/MAPK

miR-203 causes increased cell inflammation and cartilage destruction in OA rats

NF-κB

(continued)

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Table 3.1 (continued) miRNA miR204 [51] miR210 [52] miR211 [53] miR221 [53] miR222 [54] miR223 [55] miR301 [56] miR335 [57] miR338-3p [58]

Target gene

Runx2

Mesenchymal progenitor cells, double knockout mouse Bone marrow mesenchymal stem cells

HDAC4

SIRT1

Human primary chondrocytes (knee), disproportionate micromelia (DMM) mouse model ATDC5 cells

ATDC5 cells

Bone marrow mesenchymal stem cells TGF-β1

miR365 [59]

HDAC4

miR375 [60] miR411 [61]

ATG2B

miR449a [62] miR483 [63]

Cell/Animal model Human primary chondrocytes (joint), OA mouse model OA rat model

MMP-13

Fibroblast-like synovial cells, synovial samples, and knee joint effusion from patients with OA Human primary chondrocytes (joint), anterior cruciate ligament (ACL) surgery rat model OA mouse model

SIRT1, Lef-1

Human primary chondrocytes (knee), C28/I2 (human normal chondrocyte) Human mesenchymal stem cells, rat OA model

MMP-13

C57BL/6 mice

Function Anti-miR-204 treatment can save cartilage catabolism miR-210 inhibits pro-inflammatory cytokines in OA rats miR-211 is essential to maintain the homeostasis of mesenchymal joint cells Anti-miR-221 enhances cartilage formation

Signaling pathway NF-κB

NF-κB

miR-222 is involved in cartilage destruction

Upregulation of miR-223 can reduce inflammatory damage in ATDC5 cells miR-301 can alleviate the inflammatory response of ATDC5 cells There is a correlation between miR-335-5p expression and OA Downregulation of hsa-miR-338-3p promotes synovial fibrosis in patients with OA Increase of miR-365 leads to cartilage degradation

PI3K/AKT

PI3K/AKT, NF-κB Wnt/β-catenin

NF-κB

miR-375 exacerbates knee OA Overexpression of miR-411 inhibited the expression of MMP-13 miR-449a prevents cartilage degradation in OA models miR-483 may play a role in the early and late pathogenesis of OA

TGF-β

(continued)

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Table 3.1 (continued) miRNA miR486-5p [64] miR488 [65] miR558 [66]

Target gene SMAD2

Cell/Animal model Human primary chondrocytes (knee)

ZIPS

Human primary chondrocytes (knee)

COX-2

Human primary chondrocytes (knee), SW1353 cells

miR634 [67]

PIK3R1

miR675 [68]

COL2A1

Human primary chondrocytes (cartilage samples for total joint replacement surgery) Human primary chondrocytes (knee)

Function miR-486-5p inhibits the proliferation and migration of chondrocytes miR-488 protects chondrocyte differentiation/ chondrogenesis miR-558 directly targets COX-2 and regulates IL-1β-stimulated catabolism in human chondrocytes Overexpression of miR-634 may inhibit the survival rate of high-level OA chondrocytes miR-675 may be an indicator of the metabolic balance of OA

Signaling pathway

IL-lβ

NF-κB

TNF-α

For full names of abbreviations used in Table 3.1, please refer to the abbreviation list

in the development of OA by influencing the proliferation and apoptosis of chondrocytes, degenerative changes of articular cartilage, chondrocyte inflammation, and joint pain. Notably, targeting dysfunctional miRNA–mRNA interactions is an important therapeutic promise for preclinical development. This chapter introduces the relationship between miRNAs and OA to generate interest in studies exploring these aspects in OA disease pathogenesis by presenting the roles of miRNAs in OA and discusses the signaling pathways related to miRNAs in OA. The representative miRNAs which are related to OA are also summarized (Table 3.1). And the miRNAs that have potentials to become biomarkers for OA diagnosis are listed in Table 3.2. This chapter may give a critical perspective of the possible physiopathology mechanism, diagnostic and therapeutic use of miRNAs in the management of OA.

3.2

MicroRNAs and Chondrocytes

Cartilage is a type of tissue consisting only of chondrocytes, which are embedded in a collagen-rich ECM synthesized by themselves [69]. Chondrocytes derive from mesenchymal progenitor cells, can adopt different phenotypes, defined by the collagen type they produce. Despite this apparent simple composition, articular cartilages display a well-organized horizontally stratified structure, for which chondrocytes present various distribution, morphologies, and secretory profile.

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Table 3.2 miRNAs related to osteoarthritis diagnosis miRNAs miR-16 miR-132 miR-146a miR-223 miR- 210 miR-22 miR-140 miR-200c-3p miR-100-5p miR-1826 Hsa-miR-3383p miRNA-98 miR-132 miRNA-let-7e Hsa-miR-1403p Hsa-miR-6713p Hsa-miR-33b3p miR-146 a miR-155 miR-181a miR-138-5p miR-146a-5p miR-335-5p miR-9-5p miR-33a miR-136 miR-19b-3p miR-122-5p miR-486-5p

Expression Down

Position Synovial fluid

Function Diagnostic biomarkers

References [132]

Up

Synovial fluid Synovial fluid Serum

Might contribute to OA development and angiogenesis Diagnostic biomarkers

[133]

Diagnosis of KOA

[135]

Promote fibrosis of the synovial tissue Diagnosis of OA Diagnosis of OA Diagnostic biomarkers Diagnostic biomarkers

[58]

Up Down Down Down

Joint effusion Serum Serum Serum Serum

Up

PBMCs

Diagnostic biomarkers

[136, 140]

Up

OA tissues

Diagnostic biomarkers

[141]

Up

Articular cartilage Serum Serum

Regulates cholesterol synthesis

[142]

Diagnostic biomarkers Diagnostic biomarkers

[143] [144]

Up Down Down

Down

Down Up

[134]

[30] [137] [138] [139]

With OA onset, chondrocytes undergo multiple changes, in terms of not only proliferation, viability, but also secretory profile [70]. Chondrocytes are quiescent cells that rarely divide under physiological conditions [71]. Consequently, phenotypic stability, anabolic/catabolic balance activity, and survival of chondrocytes are crucial for the maintenance of proper articular cartilage [72]. Here, we will focus on chondrocyte cell death and its interplay with cartilage degradation with OA progression. Cell death of chondrocyte was reported to occur mainly through apoptosis, and autophagy, or a combination of these processes. Apoptosis is a highly regulated, active process of cell death and is a critical process in cellular homeostasis [73]. In human cartilage, apoptosis, as an initiation

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step for OA onset, comes from the observation of an increased number of apoptotic chondrocytes, or a decrease in the number of chondrocytes, or even an apoptosis-related gene perturbation within relatively macroscopically-normal OA cartilage [61]. However, it is not clear whether chondrocyte apoptosis is the inducer of cartilage degeneration or a by-product of cartilage destruction [74]. Autophagy can play a dual role in chondrocyte fate. Excessive autophagy leads to chondrocyte death and OA [75]. But there is also a view that, in some cases, autophagy constitutes a stress adaptation that avoids cell death [76]. The decline of autophagy observed during OA progression relies mainly on the hypoxia inducible factor 1 subunit alpha/hypoxia inducible factor 2 subunit alpha (HIF-1α/HIF-2α) ratio, emphasizing them as major regulators of chondrocyte survival/death, skewing the balance toward autophagy or apoptosis [77]. Many miRNAs have been proved to be a part of chondrocyte apoptosis. Chen et al. used human primary chondrocytes, rat primary chondrocytes, and SW1353 cells to demonstrate that miR-29b-3p could induce chondrocyte apoptosis and cell cycle arrest by directly targeting progranulin. Knocking down of miR-29b-3p impeded the apoptosis of articular chondrocytes and the loss of cartilage in the knee joint of surgically induced rat OA model [78]. Thus miR-29b-3p may be potential targets for OA treatment. Yang et al. found that inhibition of miR-495 suppressed chondrocyte apoptosis and promoted its proliferation through activation of the NF-κB signaling pathway by upregulation of chemokine ligand 4 (CCL4) in OA [79]. In order to explore the effect of miR-103 on chondrocyte apoptosis and its molecular mechanism in the progression of OA, Fang et al. employed OA patients and surgically induced OA rat models. They transfected miR-103 into primary rat chondrocytes and found that miR-103 reduced the expression of Sphingosine kinase-1 (SPHK1) and inhibited cell growth. In addition, the expression of total AKT and p-AKT was significantly reduced by overexpressing miR-103 in chondrocytes, while the upregulation of SPHK1 increased the expression of phosphatidylinositol 3-kinase (PI3K). They concluded that miR-103 promoted chondrocyte apoptosis by downregulation of SPHK1 and ameliorated PI3K pathway in OA [80]. Some miRNAs can regulate both apoptosis and autophagy of chondrocyte. Li et al. disclosed that overexpression of miR-766-3p inhibited chondrocyte apoptosis while promoted autophagy. On the contrary, silencing of miR-766-3p could inhibit chondrocyte autophagy and promote apoptosis. Moreover, Baicalin protects human OA chondrocytes against IL-1β-induced apoptosis and degradation by activating autophagy via miR-766-3p [81]. The death of chondrocytes in the process of OA is related to the triggering of chondrocyte autophagy and apoptosis. MicroRNA is critical for chondrocyte health, and increasing number of specific miRNAs have been suggested as regulators of chondrocyte death. Thus, miRNA has a good therapeutic prospect for the treatment of OA by protecting chondrocytes against apoptosis and modulating autophagy.

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miRNAs and the Degenerative Changes of Extracellular Matrix of Articular Cartilage

ECM is an insoluble structural component that makes up the matrix of articular cartilage [82]. It includes collagen, elastin, proteoglycans, and glycoproteins, which are secreted by chondrocytes [83, 84]. The transcription factor SOX9 (Sry-Box Transcription Factor 9) plays an essential role for chondrocyte differentiation, function and survival, and it directly regulates expression of collagen II and aggrecan [85, 86]. Chondrocyte dysfunction likely reduces ECM synthesis. On the other hand, in addition to be a major component of articular cartilage, recent studies have shown that ECM provides an important cellular environment, and cell–ECM interactions are important for regulating many biological processes, including cell growth, differentiation, and survival [87]. In articular cartilage, chondrocytes form a specific arrangement, in which chondrocytes are distributed horizontally in areas of healthy tissue [88]. During OA, chondrocytes seem to change their spatial arrangement from single to double strings and accumulate as small and large clusters [89]. The initiation and development of OA is characterized by increased production of proteolytic enzymes by chondrocytes, e.g., MMP (matrix metalloproteinase), ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), which cause the degradation of various components, and ultimately lead to cartilage damage and loss. These changes lead to alteration of the cell environment in the cartilage matrix, which acts on chondrocyte and ultimately leads to OA [90]. So, during the progression of OA, it is likely to form a vicious circle between articular chondrocyte and ECM in certain degree. The primary extraosseous chondrocytes (PEC) isolated from newborn C57BL/6 mice by Etich et al. showed elevated levels of miR-26a and adapter proteins collagen IX, matrilin-3 [91]. While, cartilage oligomeric matrix protein (COMP) and the proteolytic fragments of connective tissue growth factor (CTGF) are reduced. MiR-26a can target the hypertrophy of primary chondrocytes and the expression of adaptor protein. Therefore, the disorder of miR-26a may affect the stability of ECM in cartilage diseases [91]. The abnormal expression of miR-140 in OA cartilage derived cells as well as synovial fluid was found, indicating that the decreased level of miR-140 might be related to the progression of OA. Overexpression of miR-140 promoted the expression of collagen II in human OA chondrocytes and inhibited the expression of MMP-13 and ADAMTS-5, which were two key catabolic enzymes disposing ECM [92]. The chondrocyte numbers were also retrieved by miR-140 treatment [92]. Their results illustrated that miR-140 could alleviate OA progression by maintaining ECM homeostasis. ECM is essential to maintain the mechanical structure of articular function, as well as the homeostasis in the extracellular environment of chondrocytes. Abnormal expression of miRNAs has been suggested to link to the degradation of ECM and the degeneration of articular cartilage. Several miRNAs play a part in cartilage protection by regulation genes that mediate catabolic activity, i.e., MMP-13 and ADAMTS-5. And also miRNAs can influence chondrocyte function through ECM

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vice versa. In short, miRNAs regulate and control the process of OA through complex mechanisms, their roles in OA are still not completely clear.

3.4

miRNAs and Chondrocyte Inflammation

Chondrocytes are responsible for maintaining the normal synthesis and renewal of cartilage matrix, which may be inhibited in the inflammatory microenvironment [93]. Many literature reports showed that inflammatory mediators were parts of the pathological mechanism of OA [94]. Among these inflammatory mediators, interleukin (IL)-1β has been recognized as a promoter of the pathophysiology of OA [95]. IL-1β can stimulate chondrocyte-induced activation of the nuclear factor NF-κB pathway and lead to the expression of cartilage degrading enzymes (such as MMP-1, MMP-3, and MMP-13) [96]. In addition, IL-1β changes the expression of chondrocyte-specific proteins (including type II collagen and SOX9) in the pathogenesis of OA [97]. After inflammatory cytokine stimulation in OA, inducible nitric oxide synthase (iNOS) produces a large amount of nitric oxide resulting in overproduction of cyclooxygenase 2 (COX-2) and increased production of prostaglandin E2 (PGE2) [98]. PGE2 may increase the production of MMP-13 which leads to collagen degradation, thus IL-1β-induced inflammatory response may promote the development of OA [99]. Except chondrocytes, the inflammatory mediators also act on a variety of different cell types, including synoviocytes, osteoblasts, osteoclasts, and macrophages, thus affected the whole joints [100]. Currently, the role of inflammatory and anti-inflammatory cytokines in the pathogenesis of OA with respect to inter- and intracellular signaling pathways is still under investigation [101]. miRNA-335-5p is a miRNA that has been shown to have a significant antiinflammatory effect. Zhong et al. assessed the effect of miRNA-335-5p on the expression of inflammatory factors in human OA chondrocytes [102]. OA chondrocytes transfected with miRNA-335-5p exhibited a marked reduction in expression of the genes encoding IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α), compared with the negative control, instead cell survival and autophagyrelated factors were greatly improved. Thus miR-335-5p could significantly reduce inflammation of human OA chondrocytes by activating autophagy [102]. In addition to the protective effect of EMC and chondrocytes, miR-140-5p also possesses a status as hindering factor of inflammation. High mobility group box 1 (HMGB1), an important responder to inflammation [103], was predicted as a target of miR-140-5p. Wang et al. demonstrated that miR-140-5p was downregulated while HMGB1 was upregulated in OA [104]. MiR-140-5p could inhibit the PI3K/ AKT signaling pathway and suppress the progression of OA through targeting HMGB1. Furthermore, miR-140-5p inhibited inflammation, matrix metalloprotease expression, and apoptosis in IL-1β-induced chondrocytes through regulating HMGB1 [104]. Therefore, miR-140-5p could prevent the process of OA in at least three aspects: reducing chondrocyte apoptosis but increasing proliferation, inhibiting

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inflammation, and suppressing ECM degradation, indicating a very promising potential of miR-140-5p in OA treatment. In order to study the role of miR-197 in OA and the potential molecular mechanism, Gao et al. conducted experiments using human primary chondrocytes and found that miR-197 expression was significantly downregulated in the OA cartilage tissues compared with normal ones [105]. Furthermore, restoration of miR-197 level in chondrocytes significantly decreased IL-1β, IL-6, and TNF-α expression, whereas knocking down of miR-197 leads to the induction of these inflammatory mediators [105]. Hence miR-197 was able to inhibit inflammation in the pathogenesis of OA. Besides, the results also showed the boosting effect of miR-197 on chondrocyte growth and migration. And all these functions were achieved by targeting EIF4G2 (eukaryotic translation initiation factor 4 gamma 2), indicating the potential therapeutic targets of the miR-197/EIF4G2 axis for OA treatment [105]. The inflammatory signaling pathways involved in OA are complicated. Some different signal pathways may cause the destruction of articular cartilage by promoting the apoptosis of cartilage cells and ultimately lead to further imbalance of knee joint homeostasis. Therefore, under the cascade of inflammatory factors and signal pathways, the condition of OA will gradually worsen. Several miRNAs involved in the inflammatory response are deregulated in OA. These miRNAs might be potential targets for developing new therapeutic strategies for OA.

3.5

miRNAs and Joint Pain

OA is a prevalent whole joint disease, it is characterized by cartilage degradation, subchondral bone sclerosis and bone remodeling and synovium inflammation. All these symptoms can lead to pain [106]. So far, there is no thorough therapy for OA [107], the existing treatments are only aimed to reduce pain and swelling. The joint pain is not only the main clinical symptoms of OA, but also one of the causes of joint movement disorders in patients. Signal transduction between peripheral pain receptors and the central sensory system can cause pain. Studies have found that extracellular miR-21 can cause knee joint pain through TLR7 activation in OA rats [108]. Hoshikawa et al. performed anterior cruciate ligament transection (ACLT) on 7- to 8-week-old male rats to develop a knee joint pain model in common OA [108]. Then they injected miR-21 inhibitor into the joint cavity of the left knee after 14 days of ACLT operation. A single injection of miR-21 inhibitor or TLR7 antagonist was sufficient for long-term pain relief of knee joint, so extracellular miR-21 may be a reasonable target for the treatment of OA pain [108]. In order to solve severe joint pain and disability caused by OA, Geng et al. transfected miR-140-5p mimics and miR-140-5p expressed lentiviral plasmid into human umbilical cord mesenchymal stem cells (hUC-MSCs). They wanted to know whether hUC-MSCs influenced the proliferation of primary chondrocytes and the

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expression of cartilage genes. They performed surgery on the medial knee joint meniscus (DMM) in rats to establish the OA model and then injected hUC-MSC or hUC-MSC transfected with miR-140-5p lentivirus intra-articularly. Compared to normal hUC-MSCs, intra-articular injection of hUC-MSCs that overexpressed miR-140-5p significantly enhanced the self-repair of articular cartilage and relieved the pain [109]. To explore the effect of miR-374a-3p/WNT5B (Wingless-type MMTV integration site family member 5B) on OA, Shi et al. analyzed the proliferation of CHON001 cell after lipopolysaccharide (LPS) stimulation (an OA cell model). They indicated that the overexpression of miR-374a-3p protected CHON-001 cells from LPS attack by regulating WNT5B and inhibing JNK/ERK/MAPK pathway, which suggested the potential of miR-374a-3p for OA treatment and pain relief [110]. As the OA process intensifies, the pain will increase. Joint pain seriously affects the quality of life of patients with OA. With the deepening of the research on miRNAs, we believed that miRNAs could become a part of OA treatment and relieve joint pain. Thus, there is huge potential of applying miRNAs for pain management in OA patients.

3.6

microRNA Regulatory Pathways in OA

MiRNAs play important roles in the development of OA through many signaling pathways, including NF-κB, TGF-β, IGF, Wnt, PI3K/Akt, p53, and other pathways. These pathways mainly regulate chondrocyte metabolism (e.g., cell proliferation, differentiation, apoptosis), the synthesis/degradation of ECM, and inflammatory response processes, etc. MiRNAs could be a great part in OA therapy by mastering these regulatory pathways. The following schematic diagram shows some common signal pathways that involved in the pathogenesis of OA (Fig. 3.1).

Fig. 3.1 Signaling pathway of osteoarthritis

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3.6.1

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NF-κB Signaling Pathway

The nuclear factor NF-κB pathway is considered as a typical pro-inflammatory signaling pathway [111]. It is involved in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, oxidized LDL (low-density lipoprotein), and microbial antigens [112]. The activation of NF-κB pathway can regulate the expression of many downstream factors, such as immune proteins, cytokines, chemokines, proteases, etc. Studies have shown that OA synovium is a pro-inflammatory environment with a variety of cytokines and MMPs produced. Targeting NF-κB can diagnose and prevent OA early. For the investigation of therapeutic mechanism of miR-27a on OA, Qiu et al. used human primary chondrocytes and found that miR-27a alleviated IL-1β induced inflammatory response and articular cartilage degradation via TLR4/ NF-κB signaling pathway [113]. Using normal human articular cartilage-knee (NHAC-kn), Lei et al. revealed that overexpression of miR-382-3p reduced IL-1β induced NF-κB transcriptional activity. In addition, restoring miR-382-3p level could reduce the regulatory effect of connexin 43 (CX43) on the transcriptional activity of NF-κB. This indicated that miR-382-3p inhibited OA through NF-κB signaling pathway by targeting CX43 [114]. Dong et al. studied the antiinflammatory properties of Notoginsenoside R1 (NG-R1). ATDC5 cells were exposed to NG-R1 before LPS stimulation, and they discovered NG-R1 blocked NF-κB and renewed inflammatory damage caused by LPS by silencing miR-301a [115]. The activation of NF-κB may be an early event in the pathogenesis of OA. Its activation is a manifestation of the body’s defense response to a certain extent, but excessive activation will promote the severity of OA and the development of various complications. With the deepening of miRNA research, the OA treatment through NF-κB will continue to be improved, and it is expected that more miRNAs will be introduced to OA treatment in the future.

3.6.2

TGF-β Signaling Pathway

The transforming growth factor-β (TGF-β) signaling pathway participates in various cellular functions such as inflammation, metastasis, and embryogenesis, it can promote the differentiation of chondrogenic precursor cells and the proliferation, migration, and metabolism of chondrocytes which play an important role in OA [91]. The study of TGF-β signaling pathway will provide important basis for the development of new targets for the treatment of OA. Cao et al. treated human chondrocytes with IL-1β to induce apoptosis and cartilage degradation to reveal the function and mechanism of miR-296-5p in OA [116]. They found that overexpressed miR-296-5p activated the CTGF/p38MAPK signaling pathway by targeting TGF-β1 and reduces IL-1β-induced chondrocyte

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apoptosis and cartilage matrix degradation [116]. To test whether miR-455-3p could regulate cartilage degeneration in OA by targeting PAK2 (P21-activated kinase 2), Hu et al. used human primary chondrocytes and miR-455-3p global knockout (KO) mouse model as experimental materials and found that miR-455-3p promoted TGF-β signaling and inhibited OA development by directly targeting PAK2 [117]. Several miRNAs participate in the regulation of chondrocyte differentiation, inflammatory factors, and other physiological changes of OA through the TGF-β signaling pathway. The TGF-β pathway may be a potential target for clinical prevention and treatment of OA.

3.6.3

Wnt Signaling Pathway

Wnt signaling pathways are genetically highly conserved among animals. The Wnt signal pathway is a signal transduction pathway of a series of downstream channels stimulated by the binding of the ligand protein Wnt to the membrane protein receptor [118]. Wnt signaling represents a unique signaling pathway regulating arthritis development and progression, and there is increasing evidence that Wnts and WNT-related molecules are involved in the development and progression of osteoarthritis in human genetics and in vitro studies [119]. Wnt signaling pathways present two signaling modes between cells: intercellular communication (paracrine) or autogenous communication. During the development of OA, Wnt signaling pathway is involved in the regulation of cartilage matrix and participates in the process of bone and joint remodeling. In order to confirm whether miR-320c suppressed the development of OA by inhibiting the Wnt signaling pathway, Hu et al. evaluated the role of miR-320c in human adipose stem cells (hADSCs) [120]. They found that miR-320c decreased and β-catenin increased in the late chondrogenesis of OA chondrocytes and hADSCs [120]. In OA chondrocytes, the overexpression of miR-320c and the knockout of β-catenin have the same effects, the cartilage-specific genes are elevated while hypertrophy-related genes are downregulated [120]. In the OA mouse model, the injection of mmu-miR-320-3p attenuated the progression of OA [120]. They finally concluded that miR-320c can inhibit the degeneration of OA chondrocytes via suppressing the canonical Wnt signaling pathway [120].

3.6.4

PI3K/Akt Pathway

The phosphatidylinositol 3-kinase (PI3K)/AKT (protein kinase B) signaling pathway can participate in the regulation of many basic cell processes, including cell growth, transcription, translation, and cellular proliferation and plays an essential role in regulation of normal cell metabolism [121]. Abnormal PI3K-AKT signal transduction pathway may lead to OA.

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Tao et al. aimed to clarify the expression and function of miR-34a in OA rat [122]. Their study found that the expression of miR-34a was significantly higher in cartilage tissues and cells of OA models than that of normal tissues/cells. After miR-34a gene silencing, the apoptosis rate and the expression of apoptosis-related proteins decrease, but the cell proliferation and protein expression of the PI3K/Akt pathway increase [122]. The addition of PI3K activator also significantly promoted proliferation and inhibited apoptosis. Finally, they concluded that the downregulation of miR-34a regulated the proliferation and apoptosis of chondrocytes by activating the PI3K/Akt pathway, which provided a potential therapeutic method for the treatment of OA [122]. MiR-20 was also demonstrated as a suppressor of PI3K/AKT pathway. Inhibition of miR-20 promoted proliferation and autophagy in articular chondrocytes by activating PI3K/AKT/mTOR signaling pathway [16]. While, miR-186 could promote PI3K/AKT pathway via SPP1 (phosphoprotein 1), a regulator of PI3K/AKT pathway, and blocked chondrocyte apoptosis in mice with OA [123]. PI3K/Akt signaling pathway is involved in the regulation of cell apoptosis and is closely related to inflammatory response. Abnormal activation of PI3K/Akt can promote the high expression of apoptosis regulator Bcl-2 isoform X2 (Bcl-2) and prevent chondrocytes from undergoing apoptosis through death receptors or mitochondrial-dependent pathways. Therefore, abnormal activation of this signaling pathway is one of the key mechanisms of chondrocyte apoptosis. MiRNAs are involved in the process of OA by regulating the PI3K/Akt signaling pathway. In the future, the PI3K/Akt signaling pathway can be studied as an important target to treat OA.

3.6.5

p53 Signaling Pathway

p53 is a tumor suppressor protein that regulates the expression of a wide variety of genes, including apoptosis, growth inhibition, inhibition of cell cycle progression, differentiation and accelerated DNA repair, gene toxicity, and senescence after cell stress [124]. As tumor suppressors, p53 genes normally monitor cell division [125]. It controls the initiation of the cell cycle and participates in the process of apoptosis [126]. In order to explore the biological function and molecular mechanism of miR-3633p in chondrocyte apoptosis, Zhang et al. used a rat model of OA and rat primary cells as experimental materials to study potential of NRF1 (nuclear respiratory factor 1) to participate in OA. They suggested that miR-363-3p targeting NRF1 promoted chondrocyte apoptosis by upregulating the expression of p53 in OA rat model [127]. NAD-dependent deacetylase sirtuin-1 (SIRT1) involves in cellular aging and aging-related diseases, including OA [128]. Xu et al. studied the role of SIRT1 in epigenetically regulating p53/p21 pathway in an SIRT1 loss model [129]. They used human primary chondrocytes and primary mouse chondrocytes and observed that both chondrocyte apoptosis and p53 increased in OA progression with a declining

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expression of SIRT1 in human cartilage. And loss of SIRT1 in cartilage led to accelerated OA pathogenesis via aberrant activation of p53/p21 mediated senescence associated secretory phenotype, hypertrophy, and apoptosis. Therefore, loss of SIRT1 aggravated OA process and increases chondrocytes apoptosis by activating p53 signal pathway [129]. P53 is a tumor suppressor protein that regulates the expression of a variety of genes, including apoptosis, growth inhibition, and cell cycle inhibition. In addition, p53 can be independent of its activity and only acts as a transcription factor to trigger the apoptosis pathway. In the pathological process of OA, p53 mainly regulates OA chondrocyte apoptosis through the mitochondrial apoptosis pathway. MiRNAs can regulate the p53 pathway through a variety of pathways and ultimately participate in the development of OA. For example, miR-34a can activate p53 through SIRT1 and induce mitochondrial DNA damage, mitochondrial dysfunction, and ultimately leading to chondrocyte apoptosis. However, the research about the regulation mechanism of the above-mentioned signal pathways is not perfect. The apoptosis mechanism of p53 pathway on OA chondrocytes needs to be further studied to further clarify the pathogenesis of OA and provide reference for the prevention and treatment of OA.

3.7 3.7.1

Early Diagnosis and Treatment Using microRNAs miRNAs and Osteoarthritis Diagnosis

Early detection of OA is of vital significance for disease progression and monitoring disease response to treatment. Currently the diagnostic modalities include radiology, MRI (magnetic resonance imaging), and physical examination [130]. The clinical manifestations are varying degrees of joint swelling and pain, restricted mobility, and other symptoms. The pathological features are gradual destruction of articular cartilage and subchondral bone and inflammation of the synovial membrane of the joint. The specific blood or other body fluids (e.g., synovial fluid) testing that can be employed to aid in the diagnosis and monitoring of OA progression is still under development. Clinicians and researchers are striving for a novel biomarker(s) for early OA detection and monitoring the progression of OA. According to many studies, the biochemical changes of damaged articular cartilage precede morphological changes, so it has the ability to detect related enzymes, cytokines, and other biomarkers in body fluids. The information obtained from patients may provide new ideas for early diagnosis of OA [131]. Given the high frequency of miRNAs expression in OA, miRNAs may also play a crucial role in the early diagnosis of OA. Here we will briefly elaborate the potential of miRNAs as early diagnostic biomarkers of OA (Table 3.2). Koichi et al. measured concentrations of miR-16, miR-132, miR-146a, miR-155, and miR-223 in synovial fluid from patients with OA, and those in plasma from OA and healthy controls (HCs), and statistically examined correlations between

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miRNAs and disease activities of OA [132]. Their results supported the conclusion that synovial fluid and plasma miRNAs had potential as diagnostic biomarkers and could be used for analyses of OA pathogenesis [132]. Meanwhile, Xie et al. showed that miR-210 is significantly upregulated in synovial fluid samples of early-stage OA and late-stage OA patients compared with healthy individuals [133]. Upregulation of miR-210 in synovial fluid may occur in the early stage of OA and can be a useful biomarker for early diagnosis of OA [133]. Moreover, a research performed by Yang et al. showed that the level of miR-22 was elevated in the progression of OA [134]. While, the expression of miR-140 level in the synovial fluid was significantly reduced in the patients with OA and was negatively correlated with OA severity compared to controls [134]. Thus miR-140 and miR-22 could be considered as a potential biomarker for the diagnosis of OA [134]. In order to study the potential diagnostic value of plasma miR-200c-3p, miR-100-5p, and miR-1826 levels in knee osteoarthritis (KOA), Lai et al. measured miR-200c-3p, miR-100-5p, and miR-1826 expression levels in serum collected from 150 KOA patients and 150 healthy controls [135]. They found the diagnostic accuracy was quite high, the AUCs (area under the curve) of the diagnosis for KOA when using the serum miR-200c3p, miR-100-5p, and miR-1826 were 0.755, 0.845, and 0.749, respectively [135]. Their result indicated that the plasma levels of miR-200c-3p, miR-100-5p, and miR-1826 have potentially high value in the diagnosis of OA [135]. Soyocak et al. reported significantly higher miR-155 expression in PBMCs (peripheral mononuclear blood cells) of OA patients. And with the OA process, both miR-146a and miR-155 expression increased. Therefore, the high expression of miR-146a and miR-155 in PBMCs may be helpful for disease diagnosis and monitoring [136]. Previous studies have shown that many miRNAs are highly expressed in the synovial tissue of patients with OA, and they suggest that it is related to the pathological changes of the synovial tissue of patients with OA. And with the continuous research on OA and miRNAs, it has been found that there were many miRNA-specific expressions in the cartilage tissue of patients with OA, which suggests that we may evaluate the value of miRNAs to assist the diagnosis of OA and assessment of disease progression. Besides, as mentioned above, miRNA expression profiling in relatively non-invasive tests (e.g., using blood or synovial fluid) holds promise to reflect the disease stages or even response to therapy. The stability and relative ease of detection of miRNAs in circulation/synovial fluid show potential for diagnostic purpose. While, clearly more works need to be done in this area before miRNAs become real indicators of OA, for example, systematic evaluation of the accuracy and specificity of miRNAs as diagnostic markers and standardize the diagnosis process when using miRNAs as biomarkers, etc.

3.7.2

miRNAs and Osteoarthritis Treatment

The function of miRNAs in OA has attracted much attention in recent years. There are growing evidences that miRNAs are often dysregulated in human inflammatory

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diseases, including OA, and play crucial roles in arthritis development and joint homeostasis. Therefore, understanding of the molecular mechanisms of OA, e.g., how miRNAs influence chondrocyte numbers and cartilage degradation during OA, may help to develop new OA therapies [145]. The possibility for using miRNAs to treat OA is also discussed in Chap. 8, so here we will only briefly talk about this part. Lu et al. found miR-218-5p significantly upregulated in moderate and severe OA [146]. And functional studies showed that miR-218-5p significantly affected matrix synthesis gene expression and chondrocyte proliferation and apoptosis. It could induce cartilage destruction by regulating PI3K/Akt/mTOR axis. By using a lentiviral-based delivery system, miR-218-5p inhibitors were introduced into the joint cavity of mice and reversed the above phenomenon. Thus, the inhibition of endogenous miR-218-5p expression may be a potential method for OA therapy. Wang et al. reported that the upregulation of miR-483-5p in articular chondrocytes could delay OA cartilage destruction, because miR-483-5p directly targeted to cartilage matrix protein matrilin 3 (Matn3) and metalloproteinase 2 (Timp2), which stimulated chondrocyte hypertrophy, ECM degradation, and cartilage angiogenesis [147]. To date, there are a lot of scientists attempt to use miRNAs as treatment approach for OA. Because miRNAs participate in the regulation of OA through different physiological mechanisms, and they are probably the upstream molecular regulators of inflammatory mediators or proteinases that related to OA progression. In addition, one miRNA may regulate several genes, which may remedy the insufficient efficacy when targeting a single pathogenic factor as indicated in some clinical trials [148, 149]. The development of miRNA-based OA treatment has aroused great interest. Although the current miRNA-based treatment methods have made some progress, there are still several major difficulties that need to be resolved. First, it is essential to improve the specificity of miRNA-based treatments for bone joints to avoid unexpected adverse reactions. The good specificity of this substance is an important reason to ensure the safety of miRNA therapy. Secondly, the long-term effects of anti-miRNAs/miRNA mimics need to be further studied, which may lead to the toxicity of physiological processes. As miRNA researches become more detailed and in depth, and combined with systems biology and other research results, people will deepen their understanding of the occurrence and development of OA. With the continuous research on miRNA and OA, miRNA therapy may become an extra option besides the traditional therapies of OA in the future.

3.8

Conclusion

OA is a complicated multi-factor disease caused by not only environmental factors, but also, e.g., hormones, diet, infection, injury, and alcohol intake. Both congenital inheritance and acquired induction are related to the pathogenesis of OA. Many recent studies have shed light on the importance of miRNAs at multiple levels

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related to pathogenesis of OA. For example, advances showed that miRNAs were involved in many chondrocyte functions, such as cell proliferation, apoptosis, differentiation [8] and miRNAs also participate in changes in the ECM of articular cartilage and mediated joint pain through the control of the pathological development of OA [150]. For example, miR-140-5p was capable to slow the rate of OA by ameliorating chondrocyte functions, inhibiting inflammation, preventing ECM degradation, and relief the pain, which made it a promising therapeutic intervention in the future. Some modified stem cells which were highly expressed specific miRNA/ miRNA inhibitors, or MSC derived exosomes that are excellent sources of certain miRNAs, were also developed for OA treatment [109, 151]. In addition, these miRNAs function through the signaling pathways of many related targets, for example, NF-κB, TGF-β, IGF, Wnt, PI3K/Akt, p53, and so on. Because miRNAs can present stably in clinical samples, e.g., plasma, serum, and synovial fluid, they could be ideal biomarkers for early OA detection and for monitoring the progression of OA [104]. All these findings warrant the potential of miRNAs as therapeutic targets and or methods/indicators for OA. While, we should be aware that miRNA research in the field of OA is still in the early stages. Further studies are required to identify which miRNAs out of the bulk miRNAs reported in the literatures that have high specificity, sensitivity, and efficacy and could be applied for clinical validation in OA patients. And some other limitations also need to overcome, e.g., the delivery system, poor in vivo stability, inappropriate biodistribution, and undesirable side effects. With the continuous deepening of miRNA research, perhaps miRNA treatment will replace the current traditional treatment of OA in the future. Acknowledgement This work was supported by the Natural Science Foundation of China (82072106, 31570940, 81772017, and 81801871), the China Postdoctoral Science Foundation (2020 M683573, 2019 T120947, and 2017 M613210), the New Century Excellent Talents in University (NCET-12-0469), Shaanxi Provincial Key R&D Program (2018KWZ-10), Shaanxi Postdoctoral Science Foundation (2017BSHEDZZ13), Special Fund for Technological Innovation of Shaanxi Province (No. 2019QYPY-207), the Fundamental Research Funds for the Central Universities (3102018zy053).

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Chapter 4

Mechanosensitive MicroRNAs and Bone Formation Zhihao Chen, Yan Zhang, Ying Huai, Fan Zhao, Lifang Hu, Chaofei Yang, Ye Tian, and Airong Qian

Contents 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Mechanosensitive miRNAs and Osteogenic Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Mechanosensitive miRNAs Promoted Osteogenic Differentiation . . . . . . . . . . . . . . . . 4.2.2 Mechanosensitive miRNAs Inhibited Osteogenic Differentiation . . . . . . . . . . . . . . . . 4.3 Mechanosensitive miRNAs and Osteoblast Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Mechanosensitive miRNAs and Bone Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Mechanical stimuli are essential for maintaining bone metabolism and play key roles in regulating osteogenic cells’ differentiation or proliferation and bone formation. MicroRNAs (miRNAs) are a class of evolutionarily conserved short (~22 nucleotide fragments), single-stranded noncoding RNA involved in diverse biological processes. There are increasing number of evidences that multiple miRNAs have played a vital role in regulating osteogenesis. Significantly, recent emerging preliminary data indicate that several miRNAs (regarded as “mechanosensitive miRNA”) are sensitive to different mechanical stimuli and involved in process of bone cells activities and osteogenesis. Functional roles of mechanosensitive miRNAs in mechanotransduction during bone remodeling and further mechanism has long been a topic of scientific interest. This chapter highlights the influence of mechanosensitive miRNAs on osteogenesis and underlines their potential therapeutic applications for skeletal diseases induced by different mechanical environment.

Z. Chen · Y. Zhang · Y. Huai · F. Zhao · L. Hu · C. Yang · Y. Tian · A. Qian (*) Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, NPU-UAB Joint Laboratory for Bone Metabolism, School of Life, Northwestern Polytechnical University, Xi’an, China e-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2021 A. Qian, Y. Tian (eds.), Noncoding RNAs and Bone, https://doi.org/10.1007/978-981-16-2402-5_4

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Keywords Mechanosensitive · microRNA · Osteogenic differentiation · Osteoblast proliferation · Bone formation

Abbreviation ADSCs ALP Alp+ ATF4 BMD BMPR2 BMSCs BMP BMP2 BMP4 BV/TV cKO Col I Cav1.2 Ep300 EdU Elk1 ESW FAK FGF Fgfr2 FSS Grb2 Hmga2 LMV LTCC MACF1 MSCs miRNAs Ocn PCP Rock1 RPM Runx2 Smad4 Smad5 Smad7 TRAP

Adipose-derived mesenchymal stem cells Alkaline phosphatase Alp positive Activating transcription factor 4 Bone mineral density Bone morphogenetic protein receptor 2 Bone marrow mesenchymal stem cells Bone morphogenetic protein Bone morphogenetic protein 2 Bone morphogenetic protein 4 Bone volume/tissue volume Conditional knockout Collage I Calcium voltage-gated channel E1A binding protein p300 5-Ethynyl-20 -deoxyuridine E-twenty six like-1 Extracorporeal shockwave Focal adhesion kinase Fibroblast growth factor Fibroblast growth factor receptor 2 Fluid shear stress Growth factor receptor-bound protein 2 High mobility group AT-hook 2 Low-magnitude vibration L-type voltage-sensitive calcium channel Microtubule actin crosslinking factor 1 Mesenchymal stem cells MicroRNA Osteocalcin Planar-cell-polarity Rho associated coiled-coil containing protein kinase 1 Random positioning machine Runt related transcription factor 2 SMAD family member 4 SMAD family member 5 SMAD family member 7 tartrate-resistant acid phosphatase

4 Mechanosensitive MicroRNAs and Bone Formation

TV UTR WNT 11

4.1

81

Tissue volume Untranslated region WNT family member 11

Introduction

Bone is a specifically sensory organ that can respond to external mechanical stimuli. Exposure to mechanical unloading such as bed-rest, space flight, and hindlimb unloading or lack of mechanical forces (e.g., paralysis) led to weight-bearing bone loss and weak bone structure [1–4]. The cellular and molecular mechanisms underlying how mechanical stimuli impacted on bone remodeling have long been a topic of scientific interest. There is increasing number of evidences that multiple miRNAs serve as important regulators of osteogenesis [5–15]. Importantly, recent studies have discovered that several miRNAs were sensitive to mechanical stimulation and played a vital role in osteoblast activities and osteogenesis [7–9, 12, 16–20]. Actually, the function and mechanism of mechanosensitive miRNAs on bone formation are not yet fully understood. This chapter aims to review and analyze the role and mechanism of mechanosensitive miRNA in bone formation.

4.2

Mechanosensitive miRNAs and Osteogenic Differentiation

Because the expression of these miRNAs changes with mechanical stimulation and alters osteogenic differentiation, it is determined that mechanosensitive miRNAs participate in osteogenic differentiation. Microarray and bioinformatics analysis are effective and rapid methods to find mechanosensitive miRNAs in osteogenic cells that are changed by different mechanical stimuli. According to Wolff’s law, mechanical force is divided into hydrostatic compressive force, fluid shear stress, and mechanical stretch stress [21]. Two microarray data of MC3T3-E1 cells subjected to four-point bending stretch show that miR-3077-5p, -191*, -3103-5p, -3070a, -3090-5p are significantly increased, while miR-466i-3p, -33, -218, and -466 h-3p are decreased, accompanied by alkaline phosphatase (ALP) activity, Col I (Collage I), Alp, Ocn (osteocalcin) mRNA level and Bone morphogenetic protein 2 (BMP-2), and Bone morphogenetic protein 4 (BMP-4) protein level increased in the mechanical stretch group. In addition, analysis of putative target genes suggests that these mechanosensitive miRNAs may be involved in osteoblast differentiation [22]. MiR132-3p was up-regulated by 12% stretch in MC3T3-E1 cells, and SMAD family member 5 (Smad5) was further identified as target genes of miR-132-3p [23]. Fluid shear stress (FSS) is a common and effective type of mechanical forces in osteogenic

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cells, which promotes ALP activity, mineralization formation, and osteogenic gene expression, and is accompanied by miR-20a, -19b, -21, -34a, -200b, -140, and -34c in MC3T3-E1 osteoblastic cells [24]. The mechanosensitive miRNAs mentioned above may be involved in osteogenic differentiation. In our lab, a biaxial random positioning machine (RPM) was used to investigate mechanical unloading effects on osteoblast [25] and osteocyte [26]. In a RPM microarray data of MLO-Y4 osteocyte [26], three miRNAs including miR-15a, -29a, and -221 were decreased in osteocyte subjected to mechanical unloading environment.

4.2.1

Mechanosensitive miRNAs Promoted Osteogenic Differentiation

It is reported that a few of mechanosensitive miRNAs could promote osteogenic differentiation. The low-magnitude vibration (LMV) is also a type of mechanical loading condition, which could induce the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). The expression levels of osteogenic marker genes and miR-378a-3p were remarkably increased by LMV. Furthermore, miR-378a-3p targeted growth factor receptor-bound protein 2 (Grb2) could promote LMV-induced osteogenic differentiation of aged rat-derived BMSCs [27]. Mai et al. has shown that mechanosensitive miR-21 was decreased under FSS in MC3T3-E1 cells [24]. However, it is reported that miR-21 could directly inhibit SMAD family member 7 (Smad7) to promote osteogenic differentiation and mineralization formation in MC3T3-E1 cells [28], which indicated that miR-21 might play a different role in osteogenic differentiation under various mechanical environment. Another mechanosensitive miRNA (miR-33) was also showed the same phenomenon. The miR-33 level was down-regulated under cyclic mechanical stretch in MC3T3-E1 cells [22]. However, the miR-33-5p (one subtype of miR-33) level was up-regulated under FSS in MC3T3-E1 cells [29]. What’s more, miR-33-5p was also sensitive to mechanical unloading. To further identify the roles and mechanism of miR-33-5p in regulating osteoblast differentiation, Wang et al. investigated that loss- and gainfunction of miR-33-5p on osteoblast differentiation under FSS and mechanical unloading conditions, and high mobility group AT-hook 2 (Hmga2) which was a target of miR-33-5p and confirmed to negatively regulate osteoblast differentiation [29].

4.2.2

Mechanosensitive miRNAs Inhibited Osteogenic Differentiation

Although there are several mechanosensitive miRNAs promoting osteogenic differentiation, almost all mechanosensitive miRNAs were reported to inhibit osteogenic differentiation (Fig. 4.1). The expression of miR-29b-3p was significantly decreased

Fig. 4.1 Mechanosensitive miRNAs are involved in osteogenic cells activities and bone formation

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under four-point bending stretch by bioinformatic and real-time PCR analysis in MLO-Y4 osteocytes, whereas miR-29b-3p level in osteoblast was not changed by mechanical stretch. Moreover, the conditioned medium from osteocytes cultured 8-h under mechanical stretch could promote osteoblast differentiation. While conditioned medium from osteocytes which overexpression of miR-29b-3p repressed osteoblast differentiation [30]. The miR-154-5p level was significantly decreased under four-point bending stretch in adipose-derived mesenchymal stem cells (ADSCs). Moreover, miR-154-5p was verified to directly target Wnt family member 11 (Wnt11) to prevent ALP activity and matrix mineralization through the Wnt/planar-cell-polarity (PCP) pathway [31]. Flexcell-strain-sensitive miRNA was reported to contribute to the osteogenic differentiation of osteoblast and BMSCs [20, 32, 33]. The expression of miR-214 was down-regulated in osteoblasts after treating with mechanical strain, while overexpression of miR-214 not only inhibited the expression of osteogenic factors but also attenuated mechanical strain-enhanced osteoblast differentiation [33]. miR-103a, as a mechanosensitive miRNA, was identified to reduce hFOB 1.19 cells differentiation by directly binding to Runt related transcription factor 2 (Runx2) 3’Untranslated region (UTR) under Flexcell cyclic stretch [20]. It is reported that miR-503-5p was also stretch-sensitive miRNA, negatively regulating BMSCs differentiation [32]. In our lab, we also identified a Flexcell-sensitive miRNA, miR-138-5p, which directly targeted an important cytoskeleton protein (microtubule actin crosslinking factor 1, MACF1) to inhibit primary osteoblast differentiation [9]. Extracorporeal shockwave (ESW) is also one type of mechanical loading, which could transduce the external mechanical signals into internal biological signals to increase osteogenic differentiation [34]. In MSCs (mesenchymal stem cells) subjected to ESW treatment, the level of miR-138 was decreased. What’s more, the increased adhesion focal kinase (FAK) and Runx2 levels, and mineralization formation of MSCs induced by ESW was abrogated by overexpression of miR-138 [35]. It is reported that miR-138 targeted FAK to inhibit osteogenic differentiation of BMSCs, and miR-138 inhibitor could promote ectopic bone formation [36, 37]. Further research demonstrated that miR-138 level was markedly down-regulated in BMSCs under mechanical stretch. Moreover, miR-138 inhibitor could enhance the increase of osteogenic differentiation induced by mechanical stretch in BMSCs. In addition, miR-138 was regulated by lncRNA (H19), which functioned as a ceRNA of miR-138, and directly inhibited FAK expression to moderate BMSCs differentiation [38]. In a preceding study, miR-132-3p was up-regulated under cyclic stretch, and might regulate osteoblast differentiation by directly targeting Smad5 in response to cyclic tensile stress [23]. However, miR-132-3p level was also up-regulated and negatively correlated with primary osteoblast differentiation under mechanical unloading condition. In addition, the miR-132-3p’s target, E1A binding protein p300 (Ep300), which could inhibit the acetylation and activity of Runx2 to suppress osteoblast differentiation, and was down-regulated under mechanical unloading condition [39]. The above results also suggested that the same miRNA might play different roles in osteoblast differentiation under various mechanical stimuli. Similar

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with mechanical stretch, under mechanical unloading condition, lncRNA also directly regulated mechanosensitive miRNA to moderate osteogenic differentiation. It is reported that microgravity-sensitive miR-494 played a vital role in the BMPRSMAD-Runx2 signaling pathway through directly targeting Bone morphogenetic protein receptor 2 (Bmpr2) and Runx2 [16]. What’s more, Zhang’s group showed that Runx2-targeting mechanosensitive miR-30 family members (miR-30b, miR-30c, miR-30d, and miR-30e) were significantly increased, and negatively regulated the expression of Runx2 in MC3T3-E1 cells under mechanical unloading condition [12]. The miR-139-3p level was significantly increased in MC3T3-E1 cells under mechanical unloading condition. Furthermore, miR-139-3p was a ceRNA target of lncRNA (ODSM) and directly targeted e-26 like-1 (Elk1) to inhibit osteoblast differentiation and apoptosis in MC3T3-E1 cells [8]. We also found that miR-138-5p was mechanoresponsive to microgravity in osteoblast [9]. Taken together, mechanosensitive miRNAs acted as important regulators in osteogenic differentiation under different type of mechanical stimuli.

4.3

Mechanosensitive miRNAs and Osteoblast Proliferation

Mechanosensitive miRNAs were also reported to regulate osteoblast proliferation under different mechanical conditions. The miR-494-3p level was increased in MC3T3-E1 cells under compressive force at 294 Pa, accompanied with slower cell growth. Furthermore, miR-494-3p was identified to target Rho associated coiled-coil containing protein kinase 1 (Rock1) and fibroblast growth factor receptor 2 (Fgfr2) to negatively regulate osteoblast proliferation [40]. Osteoblast proliferation were reported to be inhibited under microgravity, Zhang’s group found that miR-103, a microgravity-sensitive miRNA [41, 42], was negatively correlated with osteoblast proliferation. What’s more, miR-103 inhibited osteoblast proliferation through targeting calcium voltage-gated channel (Cav1.2) which was a L-type voltagesensitive calcium channel (LTCC) under microgravity condition [41, 42]. Sun et al. found osteoblast cell cycle arrested in the G2 phase under simulated microgravity condition. Another microgravity-sensitive miRNA, miR-181c-5p, induced cell cycle arrest and suppressed the osteoblast proliferation via directly inhibiting cyclin B1 protein [43]. Taken together, mechanosensitive miRNAs are involved in not only osteoblast differentiation, but also osteoblast proliferation (Fig. 4.1).

4.4

Mechanosensitive miRNAs and Bone Formation

Several mechanosensitive miRNAs are reported to be involved in bone formation in vivo. Almost all reports of exercise-sensitive miRNAs in musculoskeletal system are related to skeletal muscle [44], less to skeleton. Exercise including treadmill walking, swimming and artificial cyclic strain (four-point bending on tibia), as a

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common and usefull mechanical force, is usually to explore the effects of mechanical loading on osteogenesis [45–47]. High-frequency motions [48] or daily bouts of exercise [47] were customarily considered as the prime interventions to prevent bone loss. The miR17–92 cluster was reported to play important roles in process of exercise-induced bone formation. Osteoblastic conditional knockout (cKO) miR17–92 cluster mice model producing by collagen type I promotor, showed significant decrease of bone mineral content (13–34%), maximum load (10%), and the periosteal bone formation rate (28%), while no change in the resorbing surface and bone toughness. However, compared to wild-type mice, tibia periosteal bone formation of the cKO mice revealed no obvious change after treating with two-week four-point bending exercise [49]. While, the other study suggested that inhibition of miR-92 could enhance bone volume/tissue volume (BV/TV), bone density, and tibia tissue volume (TV) by 7–16% and inhibitor negative control also showed the similar increase in these parameters (6–15%) after two-week exercise [50]. The results revealed that miR17–92 cluster is mechanosensitive miRNA cluster, which suppressed bone formation responding to mechanical exercise. It is reported that miR-222 increased in the anterior weight-bearing area of articular cartilage, which suggested that miR-222 is a potential articular cartilage mechanotransduction pathway regulator [51]. Although lack of mechanical force or physical exercise interfered with the delicate balance of the weight-bearing bone homeostasis [52]. Moreover, mechanical unloading led to the frail mechanical strength and destruction of bone microstructure, especially for the trabecular bone [53]. Previously, the mechanosensitive miR-103 family were reported to inhibit osteoblast differentiation [20] and proliferation [41, 42] in cellular level. Nevertheless, the role of the mechanosensitive miR-103 family in bone formation is still conflicting under mechanical unloading condition [20, 54]. Zuo et al. found that inhibition of miR-103a could partly counteract unloading-induced bone loss [20], but the miR-103-3p (the same as miR-142-3p and miR-30b-5p) level was down-regulated in the serum of longduration bed-rest rhesus monkeys and positively correlated to BMD [54]. Recently, miRNAs always were regarded as the potential circulating biomarkers of many diseases, due to stability of miRNAs in serum and plasma. In addition, bed-rest was generally recognized as a simulated mechanical unloading model at human level. The circulating miRNAs sequencing analysis was performed to screen for mechanosensitive miRNAs in plasma of 16 bed-rest individuals with -6 head-down for 45 days. Results showed that the expressions of miR-103, -1234, -1290, -130a, -148a, -151-3p, -151-5p, -199a-3p, -20a, -363, and -451a were significantly downregulated after treating bed-rest, and recovered after 10-day mechanical recovery, except miR-148a, -151-3p, and -199a-3p. Moreover, expression of some miRNAs among these 16 mechanosensitive miRNAs was positively correlated to bone formation, especially miR-1234 [55]. Additionally, miR-214 [5], miR-132-3p, miR-139-3p, miR-2985, miR-339-3p, miR-34b, and miR-487b [39] levels were also significantly changed in hindlimb unloading treated bone tissue. What’s more, the expression of miR-214 was increased in primary osteoblast (Alp+ cells) of femurs from four-week hindlimb unloading mice, and negatively correlated to

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bone formation [5]. Furthermore, in vitro studies showed that miR-214 inhibited osteogenic differentiation in different osteogenic cells through binding to various protein coding genes, such as activating transcription factor 4 (ATF4) in osteoblasts [5], Osterix in C2C12 myoblast cells [56], and Fibroblast growth factor (FGF) in BMSCs [57]. Shu Zhang’s Group also found that miR-132-3p [17] and miR-33-5p [7] were mechanosensitive in vivo. Before 21-day hindlimb unloading, the mice were injected with miR-132-3p inhibitor or miR-33-5p mimic by a targeted delivery system, which both effectively preserved bone loss induced by mechanical unloading [7, 17].

4.5

Conclusions

This chapter reviewed the role and mechanism of mechanosensitive miRNAs in bone formation. This chapter suggested some mechanosensitive miRNAs such as miR-103 family, miR 17-92 cluster and miR-138-5p, were identified to be respond to different mechanical stimuli and play vital roles in regulating osteogenic activities or bone formation. However, there are still some controversial roles in mechanosensitive miRNAs in regulating process of bone formation under different mechanical conditions: (1) the functions of miR-21 and miR-33 in osteogenic differentiation are opposite under stretch stress and FSS; (2) The miR-132 family level showed the same trend in osteogenic cells under mechanical loading and mechanical unloading. Furthermore, the application perspectives of the mechanosensitive miRNAs may include: (1) the potential biomarkers of disuse osteoporosis induced by mechanical condition changing in osteoporotic patients’ exosomes, serum, or plasma; (2) mimic or inhibitor of some mechanosensitive miRNAs might act as the therapeutic medicine for disuse osteoporosis by lack of mechanical force; (3) exercise-sensitive miRNAs might link crosstalk of musculoskeletal system, due to almost all exercise-sensitive miRNAs levels altering in skeletal muscles. Although the functions of the mechanosensitive miRNAs on osteogenic activities and bone formation had been reviewed in this chapter, the in-depth mechanisms of mechanosensitive miRNAs in bone remodeling under different mechanical condition remain to be lucubrated in future. This chapter will also exhibit the research findings in our lab about miR-138-5p, a mechanosensitive microRNA, after years of study [9]. We firstly identified an elevated miRNA (miR-138-5p) from either bone specimens of bedridden and aged patients with fractures or from primary osteoblasts of unloaded and aged mice, which was negatively correlated with bone formation. Secondly, we performed gain-of-function and loss-of-function of miR-138-5p to confirm that mechanosensitive miR-138-5p inhibited osteoblast differentiation under mechanical unloading or loading condition through directly targeting MACF1 by bioinformatic analysis and double dual-luciferase reporter assay. Thirdly, by genetic approaches, we constructed an osteoblast-specific miR-138-5p transgenic mice model to explore

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the role of miR-138-5p in bone formation. What’s more, we examined inhibition of osteoblast-targeted miR-138-5p could promote the mechanical bone anabolic response in miR-138-5p transgenic mice treated with treadmill exercise. Finally, by pharmacological approach, we also tested whether inhibition of osteoblastic miR-138-5p could enhance the mechanical bone anabolic response in aged mice treated with treadmill exercise or attenuate decrease of mechanical bone anabolic response in hindlimb unloading mice. We also found that miR-138-5p level was gradually decreased during osteoclast differentiation, and tartrate-resistant acid phosphatase (TRAP) activity was increased in miR-138-5p transgenic mice. Our study of mechanosensitive miR-138-5p regulating osteoblast differentiation and bone formation under different mechanical conditions may reveal a novel mechanism for osteoporosis induced by mechanical unloading and provide a potential therapeutic target for the etiology and ameliorating skeletal disorders. Acknowledgments This chapter was modified from the paper published by our group in International journal of molecular sciences (Zhihao Chen, et al. 2017; 18 (8), 1684).

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41. Sun Z, Cao X, Hu Z, Zhang L, Wang H, Zhou H, Li D, Zhang S, Xie M (2015) MiR-103 inhibits osteoblast proliferation mainly through suppressing Cav1.2 expression in simulated microgravity. Bone 76:121–128. https://doi.org/10.1016/j.bone.2015.04.006 42. Sun Z, Cao X, Zhang Z, Hu Z, Zhang L, Wang H, Zhou H, Li D, Zhang S, Xie M (2015) Simulated microgravity inhibits L-type calcium channel currents partially by the up-regulation of miR-103 in MC3T3-E1 osteoblasts. Sci Rep 5:8077. https://doi.org/10.1038/srep08077 43. Sun Z, Li Y, Wang H, Cai M, Gao S, Liu J, Tong L, Hu Z, Wang Y, Wang K, Zhang L, Cao X, Zhang S, Shi F, Zhao J (2019) miR-181c-5p mediates simulated microgravity-induced impaired osteoblast proliferation by promoting cell cycle arrested in the G2 phase. J Cell Mol Med 23 (5):3302–3316. https://doi.org/10.1111/jcmm.14220 44. Kirby TJ, McCarthy JJ (2013) MicroRNAs in skeletal muscle biology and exercise adaptation. Free Radic Biol Med 64:95–105. https://doi.org/10.1016/j.freeradbiomed.2013.07.004 45. Ju YI, Sone T, Ohnaru K, Tanaka K (1985) Fukunaga M (2015) effect of swimming exercise on three-dimensional trabecular bone microarchitecture in ovariectomized rats. J Appl Physiol 119 (9):990–997. https://doi.org/10.1152/japplphysiol.00147.2015 46. Kesavan C, Mohan S, Oberholtzer S, Wergedal JE, Baylink DJ (2005) Mechanical loadinginduced gene expression and BMD changes are different in two inbred mouse strains. J Appl Physiol 99(5):1951–1957. https://doi.org/10.1152/japplphysiol.00401.2005 47. Nogueira RC, Weeks BK, Beck BR (2014) An in-school exercise intervention to enhance bone and reduce fat in girls: the CAPO kids trial. Bone 68:92–99. https://doi.org/10.1016/j.bone. 2014.08.006 48. Ozcivici E, Garman R, Judex S (2007) High-frequency oscillatory motions enhance the simulated mechanical properties of non-weight bearing trabecular bone. J Biomech 40 (15):3404–3411. https://doi.org/10.1016/j.jbiomech.2007.05.015 49. Mohan S, Wergedal JE, Das S, Kesavan C (2015) Conditional disruption of miR17-92 cluster in collagen type I-producing osteoblasts results in reduced periosteal bone formation and bone anabolic response to exercise. Physiol Genomics 47(2):33–43. https://doi.org/10.1152/ physiolgenomics.00107.2014 50. Sengul A, Santisuk R, Xing W, Kesavan C (2013) Systemic administration of an antagomir designed to inhibit miR-92, a regulator of angiogenesis, failed to modulate skeletal anabolic response to mechanical loading. Physiol Res 62(2):221–226. https://doi.org/10.33549/ physiolres.932410 51. Dunn W, DuRaine G, Reddi AH (2009) Profiling microRNA expression in bovine articular cartilage and implications for mechanotransduction. Arthritis Rheum 60(8):2333–2339. https:// doi.org/10.1002/art.24678 52. Aguirre JI, Plotkin LI, Stewart SA, Weinstein RS, Parfitt AM, Manolagas SC, Bellido T (2006) Osteocyte apoptosis is induced by weightlessness in mice and precedes osteoclast recruitment and bone loss. J Bone Miner Res 21(4):605–615. https://doi.org/10.1359/jbmr.060107 53. Lloyd SA, Loiselle AE, Zhang Y, Donahue HJ (2013) Connexin 43 deficiency desensitizes bone to the effects of mechanical unloading through modulation of both arms of bone remodeling. Bone 57(1):76–83. https://doi.org/10.1016/j.bone.2013.07.022 54. Chen J, Li K, Pang Q, Yang C, Zhang H, Wu F, Cao H, Liu H, Wan Y, Xia W, Wang J, Dai Z, Li Y (2016) Identification of suitable reference gene and biomarkers of serum miRNAs for osteoporosis. Sci Rep 6:36347. https://doi.org/10.1038/srep36347 55. Ling S, Zhong G, Sun W, Liang F, Wu F, Li H, Li Y, Zhao D, Song J, Jin X, Wu X, Song H, Li Q, Li Y, Chen S, Xiong J, Li Y (2017) Circulating microRNAs correlated with bone loss induced by 45 days of bed rest. Front Physiol 8:69. https://doi.org/10.3389/fphys.2017.00069 56. Shi K, Lu J, Zhao Y, Wang L, Li J, Qi B, Li H, Ma C (2013) MicroRNA-214 suppresses osteogenic differentiation of C2C12 myoblast cells by targeting Osterix. Bone 55(2):487–494. https://doi.org/10.1016/j.bone.2013.04.002 57. Guo Y, Li L, Gao J, Chen X, Sang Q (2017) miR-214 suppresses the osteogenic differentiation of bone marrow-derived mesenchymal stem cells and these effects are mediated through the inhibition of the JNK and p38 pathways. Int J Mol Med 39(1):71–80. https://doi.org/10.3892/ ijmm.2016.2826

Part II

Long Noncoding RNAs and Bone

Chapter 5

Roles and Mechanism of Long Noncoding RNAs in Bone Diseases Dijie Li, Chaofei Yang, Ye Tian, Zhihao Chen, Airong Qian, and Chong Yin

Contents 5.1 Introduction of lncRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 LncRNAs and Bone Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1 LncRNAs and Osteoblasts (OBs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2 LncRNAs and Osteoclasts (OCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3 LncRNAs and Osteocytes (OCY) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.4 LncRNA in Chondrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 LncRNAs and Bone Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1 LncRNAs and Osteosarcoma (OS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2 LncRNAs and Osteoporosis (OP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3 LncRNAs and Osteoarthritis (OA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.4 LncRNA in Other Bone Related Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Mechanisms of lncRNAs Involved in Bone Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1 LncRNAs and MicroRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.2 LncRNAs Bind the mRNAs to Degrade/Stabilize Them . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.3 Natural Antisense Transcripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.4 LncRNAs and Transcription Factor/Signal Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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D. Li Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China Law Sau Fai Institute for Advancing Translational Medicine in Bone and Joint Diseases, School of Chinses Medicine, Hong Kong Baptist University, Hong Kong, SAR, China e-mail: [email protected] C. Yang · Y. Tian · Z. Chen · A. Qian · C. Yin (*) Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China e-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2021 A. Qian, Y. Tian (eds.), Noncoding RNAs and Bone, https://doi.org/10.1007/978-981-16-2402-5_5

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Abstract Close coordination of transcriptional networks and signaling pathways is necessary for the normal function of bone tissue. In pathological bone and cartilage, a lot of these networks/pathways are dysregulated. Long noncoding RNAs (lncRNAs) belong to a kind of RNAs that lack protein-coding potential. LncRNAs are usually more than 200 nucleotides length and play multifarious roles in a wide range of biological functions. In recent decades, an enormous number of lncRNAs have been identified in multiple bone cells and bone diseases and considered to play critical roles. In this chapter, we summarize the current knowledge concerning lncRNAs in bone biology and disease, from their molecular mechanism, pathological implications, and therapeutic potential. Keywords Long noncoding RNA (lncRNA) · Bone · Cartilage · Osteoblast · Osteoclast · Chondrocyte · Osteoarthritis · Osteoporosis · Osteosarcoma

Abbreviations AD AIS AMSC AS BMD BMP BMSC, BM-MSC CAVD CBF-α-1 ceRNA ChIRP circRNA CLASH CLIP DOX ECM EMSC EMT EPC EWSAT1 GO GWAS hBMSC Hh HOTAIR HSC KEGG

Adipogenic differentiation Adolescent idiopathic scoliosis Adipose-derived mesenchymal stem cell Ankylosing spondylitis Bone mineral density Bone morphogenetic protein Bone marrow-derived mesenchymal stem/stromal cells Calcific aortic valve disease Core-binding factor subunit alpha-1 Competitive endogenous RNA Chromatin isolation by RNA purifications Circular RNA Cross-linking ligation and sequencing of hybrids Cross-linking immunoprecipitation Doxorubicin Extracellular matrix Ectomesenchymal stem cells Epithelial mesenchymal transition Endothelial progenitor cell Ewing sarcoma-associated transcript 1 Gene ontology Genome-wide association study Human BMSC Hedgehog HOX antisense intergenic RNA Hematopoietic stem cell Kyoto Encyclopedia of Genes and Genomes

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lincRNA lncRNA MafB MALAT1 MEG3 miRNA MMP MNC MNPC MSC NATs ncRNAs NFAT NFATc1 NIP45 OA OC OCY OD OGS OP OPN ORF OS OVX PDLSCs POP PTBP2 PTK2 RA RNA-Seq Runx2 SFPQ TDO TF TGF-β UCA1 UTR VEGF YAP ZBED3 ZBED3-AS1

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Long intergenic noncoding RNA Long noncoding RNA v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B Metastasis-associated lung adenocarcinoma transcript 1 Maternally expressed gene 3 MicroRNA Matrix metallopeptidase Mononuclear cell Mononuclear progenitor cell Mesenchymal stem cells Natural antisense transcripts Noncoding RNAs Nuclear factor of activated T cell NFAT, cytoplasmic 1 NFAT-interacting protein 45 Osteoarthritis Osteoclast Osteocyte Osteogenic differentiation Osteogenic sarcoma Osteoporosis Osteopontin Open reading frames Osteosarcoma Ovariectomy Periodontal ligament stem cells Postmenopausal osteoporosis Polypyrimidine tract-binding protein 2 Protein tyrosine kinase 2 Rheumatoid arthritis RNA sequencing Runt-related transcription factor 2 (CBF-α-1) Splicing factor proline- and glutamine- rich Tricho-dento-osseous Transcription factor Transforming growth factor beta Urothelial carcinoma associated 1 Untranslated region Vascular endothelial growth factor Yes-associated protein Zinc finger BED-type containing 3 ZBED3 antisense RNA 1

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Introduction of lncRNA

Long noncoding RNAs (lncRNAs) are endogenous RNA transcripts that lack conserved open reading frames (ORFs) and capability to encode proteins, the length of which is usually more than 200 nucleotides (nt) [2]. LncRNAs can be categorized into several types: (1) sense lncRNAs, (2) antisense lncRNAs, (3) intergenic lncRNAs, (4) bidirectional lncRNAs, (5) intronic lncRNAs, (6) untranslated region (UTR) associated lncRNAs, and (7) promoter-associated lncRNAs [3, 4]. LncRNAs have been identified in all model organisms [5, 6]. Over 17,900 lncRNAs have been reported in recent human lncRNA annotations (GENCODE annotation for humans, v35). LncRNAs play important roles in various cell processes, e.g., chromosome dosage compensation, imprinting, transcription, epigenetic regulation, splicing, nuclear and cytoplasmic trafficking, cell cycle control, and translation [7, 8]. Moreover, interacting with proteins, DNA, or RNA via their primary sequence, secondary structures, and tertiary structures of lncRNAs, lncRNAs function in diverse ways to produce both direct and indirect profound influence on cellular processes. So far, lncRNAs have been studied a lot in many kinds of physiology and pathology processes. The expression patterns of lncRNAs are usually tissue-specific and cellspecific. Despite the initial stage of studies, lncRNAs are considered as critical players in bone biology and bone diseases. In this chapter, we summarize the current knowledge concerning lncRNAs in bone biology and disease, from their molecular mechanism, pathological implications, and therapeutic potential.

5.2

LncRNAs and Bone Cells

Generally speaking, bone cells could be mainly divided into five categories: (1) Bone marrow-derived stem/stromal cells (BMSC or BMMSC), which are multi-potent cells that have the capacity to differentiate into a variety of cell types, including osteoblasts and chondrocytes; (2) Osteoblasts (OB), which form new bone tissue; (3) Osteoclasts (OC), which pull down bone tissue; (4) Osteocytes (OCY), which hold the bone together and last as long as the organism itself; (5) Bone Lining Cells, which overlie non-remodeling bone surfaces and defend the bone; (6) Chondrocytes, which generate and maintain the cartilaginous matrix. In recent years, there are emerging reports about the way of lncRNAs regulating osteogenic differentiation (ANCR, DANCR, H19, HOTAIR, MALAT1, MEG3, etc.) or cartilage differentiation (DANCR, H19, HIT, MEG3, ZBED3-AS1, etc.) from MSCs and osteoclastic differentiation (DANCR, Neat1, TUG1, etc.) from hematopoietic stem cells (HSCs) and mononuclear progenitor cells (MNPCs). LncRNAs have been studied in various bone cells and bone-related biological processes (Fig. 5.1). A large number of studies pay attention to the osteogenesis or

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Fig. 5.1 Experimentally proved lncRNAs involving bone cells differentiation

chondrogenesis. Other cases concerning lncRNA are involved in blood cells or osteoprogenitor cells. In the blood mononuclear cells (MNCs) isolated from low bone mineral density (low-BMD) patients, lncRNA DANCR was increased and the same DANCR-induced TNF-α and IL6 in MNCs presented bone-resorbing activity [9]. In C3H10T1/2 MSCs, 116 differentially expressed lncRNAs were identified undergoing early osteoblast differentiation, such as lincRNA0231 and NR_027652 [10]. LncRNA Bmncr regulates MSC fate during skeletal aging by serving as a scaffold to facilitate the interaction of TAZ and ABL (transcription factor complex component) and subsequently helping to assemble the transcriptional complex of TAZ and RUNX2/PPARG [11].

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Table 5.1 LncRNA H19 and bone Function Promote osteogenic differentiation Promote osteogenic differentiation Promote osteogenic differentiation Promote osteogenic differentiation Promote osteogenic differentiation Promote mineralization

5.2.1

Regulation Downstream Axle miR-141-SPAG9 miR-140-5p-BMP-2/FGF9 miR-149-SDF-1 TGF-β1-Smad3-HDAC miR-138-FAK p53-NOTCH1

Ref [17] [18] [19] [12, 20] [21] [22]

LncRNAs and Osteoblasts (OBs)

Investigations of the expression and function of lncRNA during bone development and osteogenesis have provided a lot of knowledge and a deep insight into the functions of lncRNAs in osteoblast, a cell type of MSC-derived lineages. A number of lncRNAs that identified in bone are found to play a critical role during osteogenesis. A series of well-known lncRNAs, such as DANCR, H19, HOTAIR, MALAT1, and MEG3, have been extensively and thoroughly studied in other tissues. Some of those lncRNAs could promote mesenchymal stem cells (MSCs) osteogenic/ chondrogenic differentiation, such as lncRNA H19 [12], MEG3 [13], INZEB2 [14], DANCR [15], etc. Some of them could modulate the proliferation, migration, differentiation, or apoptosis of osteoblastic lineage cells. LncRNA H19, one of the first lncRNAs discovered [16], has been extensively investigated (Table 5.1). It was shown that H19 could play different roles in bone cells. In osteoblasts (OBs), H19 promotes the osteogenetic/odontogenetic differentiation of stem cells via multiple pathways, such as miR-141/SPAG9 pathway [17], miR-140-5p/BMP-2/FGF9 pathway [18], miR-149/SDF-1 axis [19], and so on. H19 positively regulates osteoblast proliferation via Wnt signaling pathway by deriving miR-675 and functioning as competing endogenous RNA for miR-22 and miR-141 [12, 20]. H19, in addition, is negatively regulated by miR-675-5p and thus modulates osteoblast proliferation [23]. In other pathway, H19 promotes miR-675 expression and contributes to the competitively binding of miR-675 to activating the Wnt/β-catenin pathway [24]. Recently, it is found that mechanical tension-induced osteogenesis of bone marrow mesenchymal stem cells (BMSCs, BM-MSCs) is mediated by H19 via FAK through sponging miR-138 [21]. H19 participates in various kinds of bone-related diseases. H19 is associated with osteoarthritis (OA) in humans [25], while it overexpresses in both OA and rheumatoid arthritis (RA) involving anabolic and catabolic conditions [26, 27]. Besides, H19 promotes mineralization by silencing NOTCH1 in calcific aortic valve disease (CAVD) [22]. LncRNA maternally expressed gene 3 (MEG3), which is firstly found to be an imprinted gene and functions as a suppressor of growth in tumor cells [28, 29], could play multiple roles in bone cells [30] (Table 5.2). In mesenchymal stem cells (MSCs) and pre-osteoblast cells, MEG3 promotes osteogenic differentiation through targeting BMP4 transcription [13] and through Wnt/β-catenin signaling pathway [38]. In periodontitis, MEG3 promotes the osteogenic differentiation of periodontal

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Table 5.2 LncRNA MEG63 and bone MSC Function Promote osteogenic differentiation Promote osteogenic differentiation Promote osteogenic differentiation Promote osteogenic differentiation Inhibit adipogenic differentiation Inhibit osteogenic differentiation Inhibit osteogenic differentiation Osteosarcoma Inhibit osteoarthritis

Regulation Downstream Axle SOX2-BMP4 miR-133a-3p-SLC39A1 miR-27a-3p-IGF1 miR-140-5p

Ref [13] [31] [32] [33]

miR-543 hNRP1-Smurf1/Runx2 hsa-miR-424 miR-16

[34] [35] [36] [37]

ligament stem cells (PDLSCs) through miR-27a-3p/IGF1 axis [32]. In human adipose-derived mesenchymal stem cells (hAMSCs), lncRNA MEG3 inhibits adipogenesis and promoted osteogenesis via miR-140-5p [33]. In BMSCs from postmenopausal osteoporosis (POP), MEG3 inhibits osteogenic differentiation by targeting miR-133a-3p [31]. However, there is limited knowledge about why lncRNA MEG3 seems to have opposite regulatory effects on MSCs osteogenic differentiation. The above findings indicate that, by modulating different targets, MEG3 could act as a switch of adipogenesis and osteogenesis. MEG3 is also found to participate in some bone diseases. In OA, MEG3 is downregulated and inversely associated with VEGF (vascular endothelial growth factor) levels [39]. In osteosarcoma (OS), MEG3, which positively regulated by lncRNA EWSAt1, facilitates the OS cell growth and metastasis and is considered as a potential predictor biomarker in both progression and poor prognosis of OS [40, 41].

5.2.2

LncRNAs and Osteoclasts (OCs)

Osteoclast (OC) is a kind of bone cells breaking down bone tissue. OCs are specialized multinucleated cells that derived from monocyte progenitor cells, macrophage progenitor cells, or hematopoietic stem cells (HSCs). Recently, emerging evidence has shown that lncRNAs could regulate osteoclastogenesis. Some of lncRNAs are found to play positive regulatory role in osteoclast differentiation. For example, lncRNA Neat1 stimulates osteoclastogenesis via sponging miR-7 [42]. LncRNA TUG1 positively regulates osteoclast differentiation by targeting MafB protein [43]. LncRNA AK077216 promotes RANKL-induced osteoclastogenesis, as well as bone resorption, via NFATc1 (nuclear factor of activated T-cells, cytoplasmic 1) by inhibition of NIP45 (nuclear factor of activated T cell (NFAT)-interacting protein) [44]. On the contrary, some lncRNAs play negative regulatory roles in osteoclast differentiation, such as Nron [45], Bmncr [46], and NONMMUT037835.2 [47].

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In OCs, the expression of lncRNAs is usually altered at different stages of osteoclastogenesis. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicate that lncRNAs play an important role in osteoimmunology [48]. In newborn and mature OCs, lncRNA AK131850 sponges miR-93-5p to enhance the secretion of VEGFa to promote vasculogenesis of endothelial progenitor cells (EPCs) during osteoclastogenesis [49]. In RA synoviocytes and OCs, lncRNA HOTAIR (HOX antisense intergenic RNA) is significantly decreased and contributes to RA pathogenesis through activating MMP-2 and MMP-13 [50]. There are some cases that lncRNAs modulate the proliferation and differentiation of OCs via canonical signaling pathway. For example, lncRNA MAYA plays roles in cancer cell-induced OC differentiation and bone resorption via activation of YAP (yes-associated protein) in Hippo-YAP pathway [51]. LncRNA linc00311 targets DLL3 to promote the proliferation and differentiation of OCs in osteoporotic rats via Notch signaling pathway [52]. With only a few lncRNAs reported, it is still not known clearly that how lncRNAs take part in osteoclast differentiation and OC-related bone disorders.

5.2.3

LncRNAs and Osteocytes (OCY)

Osteocytes (OCY), the most common cells in mature bone tissue, are terminally differentiated bone cells and can live as long as the organism itself. Osteocytes have been generally regarded to be mechanosensitive and, more to the point, a regulator of bone adaptation to mechanical loading [53, 54]. To date, there are only a few studies elucidating lncRNAs function in osteocytes. A study utilizing RNA-sequencing (RNA-Seq) analyses shows that, during the osteoblast to osteocyte transition, although the gene expression substantially changes, the majority of the transcriptome remains qualitatively osteoblast like [55]. The RNA-seq data of MLO-Y4 cells (a osteocyte-like cell line) shows that about one thousand transcripts, including a series of osteogenic markers, are changed by leptin treatment [56]. A study mapped the osteocyte-like cells (MLO-Y4 cells) response to fluid flow using RNA-Seq, in which 55 fluid flow-regulated gene transcripts are detected while lncRNAs are not mentioned [57].

5.2.4

LncRNA in Chondrocytes

Chondrocytes, which originally derive from mesenchymal stem cells, are the cells that generate and maintain the cartilaginous matrix in cartilage. It has generally known that lncRNAs regulate chondrogenic functions via affecting cartilage progenitor cells, chondrocytes, and cartilage matrix. Thousands of lncRNAs are differentially expressed in the process of chondrogenic differentiation [58]. In MSCs, lncrNA ZBED3 antisense RNA 1 (ZBED3-AS1) promotes MSC

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chondrogenesis via Wnt signal pathway [59]. DANCR, as a positive regulator to activate chondrogenesis, upregulates STAT3 and Smad3 by directly interacting with their mRNA to regulate their stability [15]. Meanwhile, DANCR could be activated by Sox4 and enhances chondrogenic differentiation and proliferation [60]. LncRNAHIT is found to be essential for chondrogenic differentiation via recruiting p100/ CBP complexes, and subsequently functions as an epigenetic regulator of chondrogenesis [61]. LncRNA H19 could regulate BMP2-induced hypertrophic differentiation of MSCs by promoting Runx2 phosphorylation [62] and attenuate force-driven cartilage degeneration via miR-483-5p/Dusp5 [63]. In chondrocytes, lncRNA HOTAIR contributes to both MMPs (matrix metallopeptidases) overexpression and chondrocyte apoptosis [64]. LncRNAs usually exhibit abnormal expression profiles in pathological process, when compared to that in the normal physiological process. In human OA chondrocytes, widespread changes of lncRNAs, such as CILinc01, CILinc02 and PACER, are associated with the inflammatory response [65]; HOTAIR upregulates and strongly promotes ADAMTS-5 expression via enhancing the mRNA stability of Adamts-5 [66]; Disordered lncRNAs (e.g., lncRNA-CIR) promote the degradation of chondrocyte extracellular matrix (ECM) [67]. In chondrosarcoma, lncRNA BCAR4 promotes the proliferation and migration by activation of mTOR signal pathway [68]. LncRNAs could affect chondrogenesis by regulating miRNA at the different stages of chondrocyte. In OA, lncRNA UFC1 sponges miR-34a to promote chondrocyte proliferation [69]. LncRNA PVT1 sponges miR-488-3p to regulate chondrocyte apoptosis [70]; LncRNA-MSR (the TMSB4 pseudogene lncRNA) competes with miRNA-152 to regulate TMSB4 expression for promoting cartilage degradation [71]. In OA-affected cartilage, lncRNA H19, along with its encoded miR-675, is significantly deregulated. In cartilage and cultured chondrocytes, H19 acts as a metabolic correlate, while the miR-675 may indirectly influence COL2A1 levels [27]. In chondrosarcoma, lncRNA HOTAIR targets Atg12 and Stat3 to increase miR-454-3p, and thus inhibiting cell growth [72]. Taken together, the above reports show that lncRNAs are involved closely in chondrocytes function, which is helpful to investigate and comprehend the regulatory mechanism of lncRNAs. Collectively, it is found that lncRNAs play an important role in almost all kinds of bone cells, most of which involving osteogenesis or chondrogenesis. Although there are also a number of cases regarding lncRNA in blood cells and osteoprogenitor cells, they are not discussed in detail in this chapter. So far, only a small number of known lncRNAs have been thoroughly identified and characterized. The future work will depend on the further identification of more lncRNAs existing/involving in bone cells. Thus, it is desirable to make global and integrated investigation of lncRNAs in bone cells, especially those involving the entire cell life span during differentiation.

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LncRNAs and Bone Diseases LncRNAs and Osteosarcoma (OS)

Osteosarcoma (OS), also named osteogenic sarcoma (OGS), is one of the most common types of cancer developing in bone. OS prefers to occur at sites where osteoblastic cells proliferate faster and prone to acquire mutations, i.e., where bone develop or grow. A series of studies reveal that there are close relationships between the lncRNAs and sensitivity/resistance of OS. 3465 lncRNAs (1704 down and 1761 up) are identified to be aberrantly expressed in doxorubicin(DOX)-resistant cell lines [73]. LncRNA CTA is found to be activated by DOX and sensitize OS cells to DOX by inhibiting cell autophagy [74]. Long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) exists in various tissues and highly expresses in many human tumors and is involved in the regulation of epigenetics and cell cycle. In osteoblastic cell, MALAT1, which can be regulated by RANKL [75], sponges miR-204 to upregulate Smad4 and thus promotes osteoblast differentiation [76]. LncRNAs, such as H19, may act as oncogenic or tumor suppressor in OS progression. Hedgehog (Hh) signaling could induce OS development via YAP1 and H19 overexpression [77]. H19 overexpresses [77] and promotes OS metastasis by acting as a ceRNA (competitive endogenous RNA) to miR-200s [78]. Loss of H19 imprinting is usually accompanied by reciprocal methylation changes at a CTCF-binding site [79]. In OS, there are a series of lncRNAs considered as autonomous prognostic biomarkers. HOTAIR is considered as a target for the diagnosis of human OS, since it is most highly expressed in OS and thus shows a correlation with advanced tumor stages, high histological grades, and poor survival rate [80]. In human OS cells, small nuclear RNA host gene 12 (SNHG12) induces the proliferation and migration of the cells by over-regulating angiomotin (AMOT) gene expression, which modulates MMP-2/MMP-9 expression [81]. BRAF-activated noncoding RNA (BANCR), which is also discovered in melanoma cells [82], shows a positive correlation with large tumor size, distant tumor metastasis, advanced clinical stage, and poor prognosis of OS [83]. Increased lncRNA UCA1 (urothelial carcinoma associated 1, UCA1) expression closely associated with distant metastasis, large tumor size, and advanced clinical stage are thus considered as an autonomous prognostic indicator for poor survival of OS [84]. There are also several circulating lncRNAs (e.g., UCA1, PARTICLE) considered as markers for poor prognosis or metastasis of OS [85, 86]. In summary, there are several lncRNA-related mechanisms in OS revealed from the above cases. A series of lncRNAs are considered as prognostic biomarkers. Some are found to be linked to the resistance to currently available chemotherapeutics. Hence, it shows great potential for developing potential diagnostic biomarkers, prognostic biomarkers, or prospective therapeutic targets that are based on lncRNAs.

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LncRNAs and Osteoporosis (OP)

Osteoporosis, a bone mass and strength concept, is a disease in which increased bone weakness results in a high risk of a broken bone. There are several risk factors of osteoporotic fracture considered as (potentially) modifiable and non-modifiable, such as age, sex, heredity, and lack of some nutrition. It has been well recognized that lncRNAs could play important role in the development of osteoporosis, although the molecular mechanism of OP has not yet been clearly clarified. Systematical analysis has utilized to profile the expression of mRNA, microRNA, and lncRNAs in ovariectomized (OVX) mice (an OP mice model) for the identification of biomarkers for OP. In a study using microarray, six differentially expressed lncRNAs are acquired from the OVX mice, including four lncRNAs that potentially function as ceRNA of miR-486-5p and miR-205-5p, respectively [87]. There are a series of lncRNAs that have been identified and considered as potential biomarker for human OP, such as lncRNA DANCR [9], XIST [88], ORLNC1 [89], and UCA1 [90]. More work is still needed to further identify the lncRNAs associated with OP and to investigate their function and mechanism.

5.3.3

LncRNAs and Osteoarthritis (OA)

OA, whose most common symptoms are joint pain and stiffness, is the most common form of arthritis worldwide. There is substantial evidence suggesting that aging is the strongest predictor for OA [91], while obesity, diet, genetics, excessive joint activity/stress, joint injuries, and inflammation are important risk factors of OA [92, 93]. Although the molecular mechanism of lncRNAs regulating OA is still not completely understood, it is widely recognized that ECM (extracellular matrix) degradation [94–96], chondrocyte apoptosis [95, 97], autophagy [98], and inflammation [99, 100] are closely related to OA. Recently, it has found that OA is highly linked to epigenetic mechanisms, in which microRNAs and lncRNAs are deeply involved [101]. It is found that, in human OA cartilage, there are a number of lncRNAs differentially expressing. Liu et al. report that, compared to that in normal cartilage, among the 30,215 coding transcripts and 33,045 lncRNAs, up to 152 lncRNAs are differentially expressed with fold change greater than 8 in OA cartilage [67, 102]. In OA cartilage, there are 3007 upregulated lncRNAs and 1707 downregulated lncRNAs, when compared to normal sample [103]. It is predicted that, in the 600 top different expressive lncRNAs, up to 530 lncRNAs might regulate their trans target genes and 48 lncRNAs have more than five cis-regulated target genes via collaboration with some transcriptional factors (TFs) [103]. The results given above shed light on the expression pattern of lncRNAs in OA cartilage, indicating that lncRNAs might also involve with the pathologic process of OA.

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In chondrocytes, numerous OA-related lncRNAs have been investigated. Many of them act as ceRNA by binding to microRNAs. LncRNA PVT1 acts as a sponge for miR-488-3p to regulate chondrocyte apoptosis [70]. UFC1 acts as a sponge for miR-34a to promote chondrocyte proliferation [69]. SNHG5 enhances chondrocyte proliferation via miR-26a/SOX2 signal axis [104]. Besides the pattern affecting proliferation and apoptosis, lncRNAs can also participate in OA through other kinds of mechanisms. Utilizing the data of long intergenic noncoding RNAs (lincRNAs) in knee/hip cartilage from OA patients, it is found that, in chondrocytes, the inflammatory response is associated with the widespread changes in the profile of lncRNAs (e.g., CILinc01, CILinc02, and PACER) [65]. LncRNA-CIR and lncRNAMSR, two newly identified lncRNAs, promote the chondrocyte ECM degradation and the development of OA [67, 71]. HOTTIP contributes to cartilage regulation by interacting with HoxA13 to modulate integrin-α1 expression and is considered as a predictive markers for OA [105]. These findings indicate that lncRNAs are deeply participated in OA pathogenesis and provide potential targets for the prevention and treatment of OA. Making further investigation on the association between lncRNAs and OA is necessary and can facilitate to find out novel potential targets for therapy.

5.3.4

LncRNA in Other Bone Related Diseases

Recent studies show that lncRNAs are involved in many other diseases. In patients with ankylosing spondylitis (AS), four lncRNA (lnc-FRG2C-3, lnc-LIN54-1, lnc-ZNF354A-1, and lnc-USP50-2) were identified, which might participate in the abnormal osteogenic differentiation of MSCs [106]. In tricho-dento-osseous (TDO) syndrome, DLX3 gene mutation interfered with bone formation by promoting BMMSC proliferation partially through H19/miR-675/NOMO1 axis [107]. The above studies reveal a role of lncRNAs in tissue-specific transcriptional regulation, providing broad and new opportunities for therapeutic strategy. In summary, there are only a small percentage of studies regarding lncRNA elucidating the function of lncRNAs in detail, most of them merely demonstrated there is a connection between lncRNAs and either bone cell biology or the development of bone diseases. Thus, it is desirable to investigate the profound relations between bone diseases and lncRNAs and further elucidate the underlying molecular mechanisms in future.

5.4

Mechanisms of lncRNAs Involved in Bone Diseases

The regulatory mechanism of lncRNAs in bone cell is complex and involves multiple types of epigenetic pattern. By virtue of the primary sequence, secondary and tertiary structures, lncRNAs function in multiple ways. The unique conformations of lncRNAs folding facilitate themselves to interact with DNA, RNA, or proteins, thus impacting on bone through regulating gene expression at transcriptional and post-transcriptional levels.

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Fig. 5.2 Paradigms of lncRNAs Mechanism involved in bone

Briefly, the main mechanism involving lncRNAs in bone cells could be divided into the following categories (Fig. 5.2): (1) Acting as a partner of chromatinmodifying complexes, which affects gene transcription, (2) Binding to messenger RNA (mRNA) via base-pairing interactions, thus allowing RNases to degrade the mRNA, (3) linking to transcription factors (TFs) that regulate bone-related gene repression or activation, (4) Acting as a scaffold for TFs and guiding the transcriptional activities across the genome in cis or trans.

5.4.1

LncRNAs and MicroRNAs

5.4.1.1

miRNAs Triggering lncRNAs to Decay

MiR-9 post-transcriptionally regulates MALAT1 RNA level in osteosarcoma cells, thus subsequently downregulates MALAT1 to promote the binding of SFPQ

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(a splicing factor factor) to Ptbp2, an oncogene inhibiting the processes of cell proliferation, migration, invasion, and epithelial mesenchymal transition (EMT) [117]. In other cases, the tumor suppressor miR-141 suppresses the proliferation but induces apoptosis of osteoblasts through downregulation of H19 via binding to H19 and inducing its translational repression [23, 118].

5.4.1.2

LncRNAs Generating miRNAs

It is found that several lncRNAs could be processed to generate miRNAs. LncRNA H19 generates miR-675 and promotes osteoblast differentiation through the H19/miR-675/TGF-β1/Smad3/HDAC pathway [12]. In the OA-affected cartilage, both H19 and its encoded miR-675 are significantly downregulated, while miR-675 subsequently influences COL2A1 levels and H19 functions as a metabolic correlate in cartilage and in cultured chondrocytes [27]. This is almost the only case of lncRNA encoding miRNA and their regulatory roles in bone cell.

5.4.1.3

lncRNAs Binding miRNAs to Derepress mRNAs

LncRNAs could sequester microRNA (miRNA) away from mRNAs, thereby derepressing mRNAs (Table 5.3). The common mechanism is that lncRNAs function as competitive endogenous RNAs (ceRNAs) (i.e., sponges for miRNAs) that transcripts share binding sites of miRNA to decrease the amounts of miRNAs that are available to target mRNAs for post-transcriptional regulation. Although there are still some controversies about the hypothesis of endogenous miRNA sponges [119], recent studies have paid great attention to this hypothesis in bone cells. LncRNA-anti-differentiation ncRNAs are shown to be important mediators of osteoblast differentiation. For example, MALAT1, having complementary bases pairing with miR-204, could upregulate Smad4 expression through inhibiting miR-204 expression and activity, and thus promote osteoblast differentiation [116]. In other cases, MALAT1 acts as a sponge of miR-206 to upregulate VEGFA expression, thereby promoting the differentiation of BMSCs into endothelial cells [120]. MALAT1 could also regulate the bone-related cell through binding or sponging a series of miRNAs, such as RNA-140-5p [121], miR-146a [122], miR-214 [123], and miR-34c [124]. LncRNA TUG1 sponges miR-204-5p to positively regulate the expression of Runx2 and thus promoting osteogenic differentiation in aortic valve calcification (calcific aortic valve disease, CAVD) [125]. H19 functions as ceRNAs for miR-29-3p and miR-200s, both of which negatively regulate the Wnt/β-catenin pathway [78, 126]. Additionally, H19 can act as a miRNA sponge for miR-141 and miR-22 to activate Wnt/β-catenin pathway and hence potentiate osteoblast differentiation. Other cases include lncRNA PVT1, MODR, MSR, etc. [70, 71, 127]. The above lncRNAs are considered as ceRNAs and contribute to various kinds of bone cells and bone diseases.

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Table 5.3 Bone-related lncRNAs showing diverse mechanisms LncRNA H19 H19 HOTAIR AK093407 Bmncr HIT MALAT1 MIR31HG ZEB1-AS1 ZFAS1

H19 DANCR

circRNAvgll3 Neat1 MALAT1 MSR

Role Mechanism Chromatin remodeling Chromatin modifier Methylate DNA Chromatin modifier Histone deacetylase Chromatin modifier Methylate DNA Transcriptional activation/suppression Interact with TF Phosphorylation Scaffold Facilitate TFs assembly Enhancer Recruit TF complexes Interact with TF Recruit TF Bind to protein Phosphorylation Enhancer Recruit TF Interact with TF Post-transcriptional modification Bind to protein Control the decay of mRNA Interact with Regulate mRNA mRNA stability Interact with microRNA microRNA sponge miR-326-5p microRNA sponge microRNA sponge

miR-7, PTK2 microRNA

Interact with microRNAs

Decay lncRNAs

(Target)/Cell function

Ref

(Notch1), mineralization Adipocyte differentiation Chondrosarcoma growth

[22] [108] [72]

Proliferation, apoptosis Osteogenesis

[109] [11]

Chondrogenesis (SP3, LTBP3), myeloma (IκBα, NF-κB) (ZEB1) oncogenesis (ZEB2) osteosarcoma metastasis

[61] [110] [111] [112] [113]

Osteoblastic differentiation Chondrogenic differentiation

[114]

Osteogenic differentiation Osteoclastogenesis Osteogenesis/ chondrogenesis Cartilage degradation

[15]

[115] [42] [116] [71]

In articular chondrocytes, it is found that MALAT1 suppresses miR-127-5p expression through directly targeting miR-127-5p, as well as miR-127-5p directly binds to the 30 UTR of Osteopontin (OPN) to suppress its expression, thus subsequently regulating the proliferation of chondrocytes via PI3K/Akt pathway [128]. In MSCs, H19 regulates BMP2-mediated hypertrophic differentiation via promoting the phosphorylation of Runx2 [62]. In chondrocytes, H19 promotes the proliferation and migration of chondrocytes and suppresses the degradation of matrix possibly by targeting the miR-106b-5p/TIMP2 axis [129]. The lncRNA–miRNA interactions identified to date are summarized in Table 5.4. Although it has gained attention, the essential relationship between lncRNAs and miRNAs is still necessary to elucidate for better therapeutic strategies.

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Table 5.4 LncRNAs and their target miRNA involved in bone cells Regulation Axle MODR-miR-454-Runx2 MALAT1-miR-204-Smad4 MALAT1-miR-214-ATF4 MALAT1-miR-320a-5p-Runx2 MALAT1-miR-146a-PI3K/Akt/ mTOR MALAT1-miR-202 TUG1-miR-204-5p-Runx2 TUG1-miR-212-3p-FOXA1 TUG1-miR-9-5p-POU2F TUG1-miR-132-3p-SOX4 TUG1-miR-143-5p-HIF-1α TUG1-miR-335-5p-ROCK1 H19-miR-141-Wnt H19-miR-22-Wnt H19-miR-140-5p-BMP-2/FGF9 H19-miR-29b-3p-SOX9 H19-FoxO3 H19-miR-483-5p-Dusp5 H19-miR-200s-ZEB1/ZEB2 PVT1-miR-146a-TGF-β/SMAD4 PVT1-miR-488-3p PVT1-miR-195-BCL2/CCND1/ FASN miR20HG-miR-503XIST-miR-19a-3p-Hoxa5 XIST-miR-21-5p-PDCD 4 ZEB1-AS1-miR-200s-ZEB1 SNHG15-miR-141-MMP2/MMP9 SNHG16-miR-485-5p-BMP7 MSR-miR-152-TMSB4 CIR-miR-27b-MMP13

Target cell MSC Osteoblast MSC Osteogenic cell Chondrocyte

Function Differentiation Differentiation Differentiation Differentiation Proliferation

Ref [127] [116] [123] [130] [122]

Osteosarcoma cell Osteoblast Osteosarcoma cell Osteosarcoma cell Osteosarcoma cell Osteosarcoma cell Osteosarcoma cell Osteoblast

Metastasis

[131]

Differentiation Proliferation and apoptosis

[125] [132]

Tumorigenesis

[133]

Proliferation and apoptosis

[134]

Metastasis

[135]

Migration and invasion

[136]

Differentiation

[20]

Differentiation Differentiation Osteochondral activity Degeneration Metastasis

[18] [137] [138] [63] [78]

Degradation Apoptosis Development

[139] [70] [140]

Invasion and metastasis

[141]

Differentiation Growth and metastasis

[142] [143]

Proliferation and migration

[144]

Proliferation, invasion, autophagy Differentiation Cartilage degradation Cartilage degradation

[145]

Odontoblastic Chondrocyte Chondrocyte Chondrocyte Osteosarcoma cell Chondrocyte Chondrocyte Osteosarcoma cell Osteosarcoma cell Osteoblast Osteosarcoma cell Osteosarcoma cell Osteosarcoma cell BMSC Chondrocyte Chondrocyte

[146] [71] [147]

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5.4.2

111

LncRNAs Bind the mRNAs to Degrade/Stabilize Them

So far, it has been demonstrated that the interaction between lncRNAs and mRNAs could upregulate the mRNAs by stabilizing them or, on the contrary, downregulate the mRNA by allowing RNase to degrade them. LncRNA DANCR can directly interact with the mRNA of Myc, Smad3, and STAT3 to control their stability and elongate their half-life, thus subsequently activating synovium-derived MSCs chondrogenesis through upregulation of Smad3 and STAT3 expression [15]. Although in several cases the detailed mechanism of lncRNA regulating mRNA is still unclearly known, lncRNAs strongly promote their target mRNA expression by increasing the mRNA stability which is a common mechanism. For example, HOTAIR promotes ADAMTS-5 in OA articular chondrocytes [66] and AS-IFITM5 increases IFITM5 in osteoblasts [148]. The reciprocal relationship between mRNA-lncRNA pairs seems to be a common mechanism, especially in natural antisense transcripts (NATs), one kind of lncRNAs discussed in the next section.

5.4.3

Natural Antisense Transcripts

NATs are naturally occurring RNA transcripts, which are complementary to endogenous RNA transcripts [149]. The regulation mechanisms of NATs include chromatin remodeling, transcriptional interference, RNA splicing, RNA editing, RNA masking, and so on. Emerging bone-related NATs have been found to inhibit/promote/switch the critical physiological events of bone formation, since Msx1os, the antisense (AS) mRNA of Msx1 (Msx1-AS RNA) was firstly identified in 2002 from mouse dental and bone cells [150, 151]. LncRNA AS-IFITM5 increases sense IFITM5 transcription to enhance bone formation [148]. LncRNA FGFR3-AS1 regulates its NATs, FGFR3, to promote osteosarcoma growth [152]. There have been several reports regarding NATs in bone and skeletal disease. In a genome-wide association study (GWAS) for adolescent idiopathic scoliosis (AIS) in Chinese girls from a sample of 4317 AIS patients, new susceptibility loci and 6016 controls are identified. LBX1AS1, an antisense transcript of LBX1, is considered to be a functional variant of AIS and contributes to the risk of AIS [153]. Devoid of Dlx1 antisense RNA (Dlx1-AS) displays a mild skeletal phenotype reminiscent of a Dlx1 gain-of function phenotype. Dlx1-AS impacts on the life span of Dlx1 through decaying of the sense transcript, thus modulating Dlx1 transcript levels and availability [154]. Recently, increasing number of investigations paid attention to the NATs previously reported in other tissues. In osteogenic differentiation, NATs are found to play important role. LncRNA HoxA-AS3, which is required for H3K27me3 of the key osteogenic transcription factor Runx2, functions as an epigenetic switch to

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determine the lineage specification of MSCs through interacting with EZH2 [155]. LncRNA HIF-1α-AS1, which is widely expressed in tumor tissues [156, 157], could be induced by TGF-β and thus promote osteoblastic differentiation of hBMSCs [158]. Recently, the antisense RNA of HIF-1α, HIF-1α-AS1, and HIF-1α-AS2 regulates HIF-1α and thus affecting the osteogenic differentiation of PDLCs under hypoxia. Both HIF-1α-AS1 and HIF-1α-AS2 are complementary to HIF-1α mRNA. HIF-1α has positive regulatory effects on HIF-1α-AS1 and HIF-1α-AS2, while only HIF-1α-AS2 shows an inhibitory effect on HIF-1α [159]. There are several NATs differentially expressed in osteosarcoma (OS) and considering to playing important roles in OS. The expression profile of 25,733 lncRNAs in osteosarcomas (OS) compared with paired adjacent non-cancerous tissue has been described, in which SATB2-AS1, a novel antisense long noncoding RNA, is identified and found to be significantly overexpressed [160]. It firstly documents that, in OS patients and in human OS cancer cell lines (HOS, MG63, U2OS), SATB2-AS1 is upregulated, which promotes the proliferation and growth of the cells via regulating the expression of its sense gene SATB2 [160]. Two NATs, FOXC2-AS1 and Wrap531α, are overexpressed in OS and regulate their NATs, respectively, thus promoting doxorubicin resistance or growth of OS [161, 162].

5.4.4

LncRNAs and Transcription Factor/Signal Pathways

Normal bone development requires tight coordination of signaling pathways and transcriptional networks. Many of these signaling pathways are dysregulated under pathological conditions that affect cartilage and bone. Emerging lncRNAs have been identified to participate in the development or maintaining of cellular phenotypes in cartilage and bone. There are many signaling pathways involved in MSCs, osteoblastic lineage cells (Fig. 5.3), osteoclastic lineage cells, and chondrocytes, etc. (Table 5.5). Several pathways are recognized to be associated with osteogenic differentiation potentials of BMSCs, including Wnt signal pathway, BMP signal pathway, TGF-β signal pathway, MAPK signal pathway and Toll-like receptor signal pathway, etc. Utilizing human whole transcriptome microarray, the expression profiles of differential lncRNAs are identified and functional network is analyzed, which reveals that 13 core regulatory genes including six lncRNAs (FR374455, XR_ 111050, NR_ 024031, FR406817, FR148647, and FR401275) are involved in the above pathways [182]. Utilizing RNA-Seq, there are lots of lncRNAs identified to play role in osteogenic differentiation of MSCs [183]. These lncRNAs regulate the interaction of ECM-receptor and focal adhesion pathways by targeting their co-expressed genes (WNT2, COL21A1, and COL4A4) [183]. The Wnt/β-catenin is crucial during bone development and is involved in BMSCs, osteoblasts, osteocytes, and chondrocytes [184–186]. LncRNAs, such as DANCR and ANCR, suppress osteoblastic differentiation through inhibiting Wnt/β-catenin pathway. On the contrary, lncRNAs, such as H19 and lnc-POIR,

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Fig. 5.3 The effect of lncRNAs on signaling pathway in osteoblastic lineage cells

promote the osteoblast differentiation through activating or up-regulating Wnt/β-catenin pathway [20, 187]. H19 can also accelerate the osteogenic program through altering the NOTCH1 pathway in CAVD [22]. In addition, the association of Wnt/β-catenin pathway has widely reported in different bone cancers [188]. There are various different mechanisms of lncRNAs regulating or being modulated by bone-related signal pathway. Some studies reveal that lncRNAs alter gene

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Table 5.5 LncRNAs and related signaling pathway involved in bone Action Promote

Pathway Wnt/β-catenin

lncRNA H19

Target Cell Type MSC

Inhibit

Wnt/β-catenin

H19

MSC

Inhibit

Wnt/β-catenin

ANCR

hPDLSCs

Inhibit

Wnt/β-catenin

DANCR

BMSC

Inhibit

Wnt/β-catenin

DANCR

DPCs

Inhibit Promote

Wnt/β-catenin Wnt/β-catenin

LET SNHG1

BMSC OS

Promote

Wnt/β-catenin

SOX4

OS

Inhibit

Wnt/β-catenin

NEAT1

OS

Promote

Wnt/β-catenin

OS

Promote Promote

Wnt/β-catenin Wnt/β-catenin

Inhibit

Wnt & JNK

HNF1AAS1 HOTTIP MALAT1 TGU1 MEG3

OS

Promote

BMP

MEG3

BMSC

Promote

BMP/Smad

INZEB2

BMSC

Inhibit Promote

LET HOTAIR

BMSC Chondrocyte

Inhibit

TGF-β MAPK1/ c-Jun p38 MAPK

DANCR

BMSC

Inhibit

Notch

MEG3

OS

Promote

Notch

H19

MSC

Promote

Notch

SNHG12

OS

Promote Promote

NF-κB FAK

HOTAIR H19

Chondrocyte BMSC

Promote

CXCL13

AK028326

BMSC

Inhibit

AKT

MEG3

Synoviocyte

OS OS

Function Promote osteoblast differentiation Promote osteogenic differentiation Promote osteogenic differentiation Inhibit osteogenic differentiation Inhibit odontoblast-like differentiation Inhibit proliferation Promote osteosarcoma progression Promote osteosarcoma progression Promote osteosarcoma progression Promote osteosarcoma progression Inhibit chemoresistance Promote osteosarcoma progression Inhibit osteosarcoma progression Promote osteogenic differentiation Promote osteogenic differentiation Inhibit proliferation Promote inflammation Promote proliferation and osteogenic differentiation Inhibit osteosarcoma progression Promote osteogenic differentiation Promote osteosarcoma progression Promote inflammation Promote osteogenic differentiation Promote osteogenic differentiation Promote proliferation

Ref [20] [163] [164] [165] [166] [167] [168] [169] [170] [171]

[172] [173] [13] [14] [167] [174] [175] [176] [177] [178] [174] [21] [179] [180] (continued)

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Table 5.5 (continued) Action Promote

Pathway AKT

lncRNA MALAT1

Promote

Hippo-YAP

MAYA

Target Cell Type OS Breast cancer cell, OC

Function Promote osteosarcoma progression Promote bone metastasis Promote osteoclast differentiation

Ref [181] [51]

activity by interacting with the chromatin or DNA. There are many lncRNAs that can directly bind, the region or nearby, the transcription initiation site of their target genes. MEG3 and BCAR4 activate the promoter region of BMP4 and GLI2, respectively [13, 189]. H19 modulates histone deacetylases to inhibit BMSCs adipocyte differentiation through a CTCF/H19/miR-675/HDAC regulatory pathway [108]. By interacting with protein factor, lncRNA can repress or activate signaling pathway. MALAT1 directly interacts with LTBP3 and SP1, thus modulating the recruitment of Sp1 to the LTBP3 gene that regulates the TGF-β bioavailability in MSC from multiple myeloma [110]. Briefly, emerging evidence show that lncRNAs could be major regulators of cell signaling pathway and essential for various fundamental cellular processes involved in bone disease, mainly via recruitment of different proteins or RNAs to DNA/RNA-protein complexes. It is necessary to make further investigation of signal pathway and their mechanisms in bone-associated diseases.

5.5

Conclusions and Perspectives

Over the past two decades, extensive researches have showed that lncRNAs could play important roles in diverse bone-related cellular processes. In different pathogenesis of bone diseases, the molecular mechanisms of lncRNAs show a variety of individuation multiplicity. The profiles of lncRNAs are cell-specific in bone. There are a large number of lncRNAs that are identified in bone cells. For further validation, it is also a task to identify functional lncRNAs from hundreds and thousands of candidates. On the other hand, experimental evidences highlight that lncRNAs could be potential biomarkers for diagnosis, therapy, and prognosis in patients with bone diseases, especially bone cancer. However, the research studies based on therapeutic application of lncRNAs for bone diseases are still on the way. In this chapter, we summarized a series of newly or classically discovered lncRNAs and their mechanism in a variety of bone cells (i.e., osteoblasts, osteoclasts, osteocytes, and chondrocytes) and bone disease. It is universally known that lncRNAs play their roles mainly by interacting with other molecules, such as DNA, protein, microRNA, mRNA, or even another lncRNA. However, compared to mRNA and small RNA, the current knowledge of lncRNAs is still at an early

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stage and many questions remain unanswered to date. For example, although emerging evidences suggest that lncRNAs could function as critical regulators in different kinds of bone cells, the role of lncRNAs in bone development and many bone diseases is not clear. As far as we know, lncRNAs have almost yet to be investigated in early bone development. This might possibly be due to the detection problems, such as that the half-life of lncRNA is usually obviously shorter compared to mRNA and, in the past, the identification of skeletal stem cells is ambiguous. The development of novel technologies, such as high-throughput sequencing, CLIP (cross-linking immunoprecipitation) [190, 191], RNA pulldown, CLASH (crosslinking ligation and sequencing of hybrids) [192, 193], and ChIRP (chromatin isolation by RNA purifications) [194, 195], has also provided new tools to investigate lncRNAs and their networks involving other molecules. In summary, the functions and regulatory mechanism of bone-related lncRNAs remain to be elucidated. Most of what we know is still limited to cellular levels. At present, how lncRNAs potentially regulate bone development and homeostasis remains unclear. In this field, research interests might dramatically increase over the coming years and provide people with critical knowledge on new lncRNAs involving bone. Thus, it might have deep impacts on the understanding of bonerelated diseases, such as osteosarcoma, osteoporosis, osteoarthritis, and so on, and will provide new strategies for their diagnosis, therapy, and prognosis. Acknowledgements This chapter was modified from the paper published by our group in Endocrine Metabolic & Immune Disorders-Drug Targets (Li DJ, et.al., 2020, 20 (1), 50–66). The authors would like to thank Ge Zhang and Jin Liu at the Hong Kong Baptist University for their generous indispensable support and constructive suggestions. This work was supported by the Natural Science Foundation of China (82072106, 81801871, and 81772017), the China Postdoctoral Science Foundation (2020M683573 and 2018T111099), the Innovation Foundation for Doctor Dissertation of Northwestern Polytechnical University (CX201821).

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Chapter 6

Long Noncoding RNAs Regulate Osteoblast Function and Bone Formation Chong Yin, Ye Tian, Xuechao Liang, Dijie Li, Shanfeng Jiang, Xue Wang, and Airong Qian

Contents 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Obstacle on Osteogenic lncRNA Researches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Screening of Osteogenic lncRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Osteogenic lncRNA Researches In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Mechanism Researches of Osteogenic lncRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6 Osteogenic lncRNA as Endogenous miRNA Sponge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Long noncoding (lnc) RNAs play an essential role on bone formation. Increasing evidences have proved that lncRNAs may be adopted as potential therapeutic targets for osteoporosis. However, some problems still exist in the study of osteogenic lncRNAs. The screening of lncRNAs related to osteogenic differentiation has been proved expensive and low efficient, the functional study of the lncRNA in vivo was very hard to establish, it was difficult to study mechanism of lncRNAs,

C. Yin · Y. Tian · X. Liang · S. Jiang · X. Wang · A. Qian (*) Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China e-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected] D. Li Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China Law Sau Fai Institute for Advancing Translational Medicine in Bone and Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, SAR, China e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2021 A. Qian, Y. Tian (eds.), Noncoding RNAs and Bone, https://doi.org/10.1007/978-981-16-2402-5_6

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and the low homology of lncRNAs between different species made lncRNA study hard to be used as therapeutic targets for osteoporosis. This chapter will briefly summarize the defects in lncRNA today, and describe how we solve this problem of lncRNAs in our previous studies. We have developed some methods that would significantly enhance the efficiency and reduce the cost of lncRNA researches. We hope these methods would make osteogenic lncRNA study simplified and thus made osteogenic lncRNAs closer to a therapeutic target for osteoporosis. Keywords Long noncoding RNA · Osteoblast differentiation · Bone formation · Osteoporosis · Endogenous miRNA sponge

Abbreviations BMSCs ceRNA HLU KEGG LncRNAs miRNA MSCs ncRNAs OP OVX PCC RT-PCR

6.1

Bone marrow mesenchymal stem cells Competitive endogenous RNA Hind limb unloading Kyoto encyclopedia of genes and genomes Long noncoding RNAs microRNA Mesenchymal stem cells Noncoding RNAs Osteoporosis Ovariectomized Pearson’s correlation coefficient Real-time polymerase chain reaction

Introduction

LncRNAs have been proved as an essential part on regulating osteoblast differentiation and bone formation. Researches on multiple lncRNAs regulating osteogenic differentiation have been reported by many researchers previously. For example, Wang et al. showed that lncRNA ODSM promoted osteoblast function and bone formation through miR-139-3p/ELK1 axis [1, 2]. Li et al. revealed that lncRNA H19 promoted bone formation by activating Erk and Wnt/β-catenin signaling pathway [3, 4]. Feng et al. found lncRNA XIST sponged miR-214-3p to promote osteogenic differentiation of periodontal ligament stem cells [5]. Mulati et al. established lncRNA Crnde knock-out mice, and found Crnde significantly enhanced Wnt/β-catenin signaling pathway and further enhanced bone formation [6]. Zhang et al. revealed that lncRNA NEAT1 promoted osteogenic differentiation of human bone marrow-derived mesenchymal stem cells through NEAT1/miR-29b-3p/BMP1

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axis [7]. He et al. discovered a novel osteogenic lncRNA ODIR1 which interacted with F-box protein 25 and inhibited the osteogenic differentiation through the FBXO25/H2BK120ub/H3K4me3/OSX axis [8]. These studies supplemented the physiological mechanisms of bone formation, and also suggested that the identification of lncRNAs would provide more potential therapeutic targets for rescuing osteoporosis.

6.2

Obstacle on Osteogenic lncRNA Researches

However, some problems still exist in the osteogenic lncRNA research today. Firstly, the screening of lncRNA is difficult and unreliable. Screening of lncRNAs usually relays on massive RNA-seq assay, which is proved quite expensive. Moreover, finding out osteogenic lncRNAs is very hard from the RNA-seq result. Secondly, in vivo experiments of the osteogenic lncRNAs are difficult to establish. Although lncRNAs can be silenced or over-expressed by siRNAs or vectors, manipulating the expression of lncRNAs requires techniques to deliver the siRNAs or vectors directly to bone formation surface, which requires very high cost for nucleic acid purchasing and sophisticated experimental operation. Thirdly, the mechanisms of lncRNAs are multiple and complicated. LncRNAs might regulate bone formation with different pathways or even different mechanisms, and how to figure out an essential mechanism for a lncRNA is labor intensive. Moreover, the homology of lncRNAs between different species is low, which means that the lncRNAs screened and functional tested in mice or rats could not be used on human. That made problems for therapeutic application of the lncRNAs. For lncRNAs interacting with proteins, the domain that bond with protein might not maintain its bonding function in different species. As for lncRNAs work as ceRNAs, the bonding site would be easier to be preserved, but its regulating efficiency is still highly various between different species. Our research group had established a system for screening and functional analysis of lncRNAs, which was proved high efficiency and would overcome these problems. Here we will briefly describe this system based on our lncRNA results and provide ideas for osteogenic lncRNA researchers in their studies.

6.3

Screening of Osteogenic lncRNAs

Our research group has discovered a method that could systematically screen osteogenic related lncRNAs. In our study, we adopted a special cell model, named as MACF1-knockdown MC3T3-E1 cell which presented a reduced osteoblastic differentiation rate [9]. In our study, this MACF1-knockdown MC3T3-E1 cell and its negative control were tested by mRNA-array and lncRNA-array. In further

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studies, osteoblast or mesenchymal stem cells with different period of osteogenic differentiation can also be adopted for the screening. The lncRNA-array results may contain hundreds or thousands of lncRNAs, and these lncRNAs were screened by certain standards. Firstly, lncRNAs with length more than 3500 nt might be hard to insert into vectors and transfect into cells, while lncRNAs that shorter than 800 nt might be hard to design siRNAs, which made it unable to determine its function. Thus, we only select lncRNAs with length between 800 and 3500 nt. Secondly, we select lncRNAs that highly correlated with osteogenic differentiation, and we only chose lncRNAs that were significantly changed in MACF1-knockdown MC3T3-E1 cell. We than designed RT-PCR primers for all lncRNAs with 800–3500 nt in length and significantly changed in array data. RT-PCR was established in normal MC3T3-E1 cells first, and lncRNAs with a CT value higher than 42 was considered as very low expressed in osteoblast, indicating negligible role in osteogenic differentiation, and these lncRNAs were weeded out. The remaining lncRNAs were selected for further screening. Besides, KEGG clustering analysis was used to evaluate osteogenic genes that were affected within the mRNA-array. All significantly changed mRNAs which associated with osteogenic signaling pathways (including Wnt, BMP, TGF-beta, and HIF-1 signaling pathway), along with essential osteogenic marker genes were selected and a co-expression network of selected lncRNAs and osteogenic mRNAs was established by calculating Pearson's correlation coefficient (PCC). The average co-expression level of each lncRNAs was listed and ranked by absolute value. 25 lncRNAs with highest absolute value of average co-expression level were considered as highly correlated with osteogenic differentiation. Then, the expression patterns of these 25 lncRNAs were investigated in bone marrow mesenchymal stem cells (BMSCs) isolated from multiple osteoporosis mice models, including aging mice, ovariectomized (OVX) menopause mice, and hind limb unloading (HLU) osteoporosis mice. LncRNAs that significantly changed with the same pattern in all three mice models were selected for further testing. Next, correlation analysis was proceeded for the expression pattern in mice. In brief, the expression levels of all lncRNAs, multiple osteogenic marker genes, and transcription factors were detected by RT-PCR in the femur tissue of different ages of C57BL/6 mice. A correlation analysis was made between each lncRNA and each osteogenic mRNAs and the results revealed the correlation ship with osteogenic differentiation of each lncRNA.

6.4

Osteogenic lncRNA Researches In Vivo

In vivo studies of osteogenic lncRNAs are much more arduous than in vitro. For in vitro studies, expression level of lncRNAs can be manipulated by transfecting siRNAs or vectors into cultural osteoblast or mesenchymal stem cells. But alteration of lncRNAs in vivo is not easy. The transfection reagent needs to have the capacity that directly delivers the siRNAs or vectors to bone formation surface, and also it

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should maintain on this bone formation surface for at least 4 h to make the in vivo transfection efficiently functional. Moreover, the cost of both transfection reagent and the nucleic acid is very high. So far, only a few studies reported the function of lncRNA in bone formation, in our previous studies, siRNAs of AK016739 and AK045490 were injected subcutaneously over the calvarial surface [10, 11]. This injection could keep the transfection reagent carrying the nucleic acid enough time on the bone formation surface of mice calvaria, while the injection only affects on a small area of mice calvaria, so the volume and amount of transfection reagent and the nucleic acid were highly reduced. One month after this transfection, calvaria was labeled by calcein, and the calvaria was embedded in OCT without decalcification and dissected by 4μm in thickness. Slides were examined with a fluorescent microscope and bone dynamic histomorphometric analyses for mineral apposition rate were performed. This method could clearly reveal the bone formation of mice treated within 30 days. Along with reduced cost and simplified techniques, which could greatly reduce the time and reagent cost for the in vivo study of osteogenic lncRNAs. However, aging and postmenopausal osteoporosis mostly occur in weightbearing bones, especially femur and tibia, which made it impossible to do in vivo experiments on calvaria. In Yuan’s study, siRNA was administrated into the bone marrow of mice femur to investigate the function of PGC1β-OT1 on femoral bone formation [12]. Our research group also use overexpression plasmids or siRNAs to change the expression of lncRNAs (e.g., lnc-DIF and AK018451) by injection them into medullary cavity of femur (unpublished results). The identification of the bone formation alteration can be made by microCT and calcein labeling. RNA based therapy has become an emerging tendency in exploring new methods to rescue bone metabolic diseases. As we have known that lncRNAs can also be a potential therapeutic target. While delivering the nucleic acid precisely to target osteoblasts is essentially required. The development of novel RNA delivery system with a bone specific aptamer would transport RNA sequences to its target region with high efficiency [13–16]. The technique had made the siRNA or vectors of lncRNAs as a potential therapy of osteoporosis. In our study, we have investigated the rescue effect of si-lnc-DIF to postmenopausal osteoporosis by intravenous injecting si-lnc-DIF carried by an osteoblast-targeting delivery system. The results proved that this precise delivery of si-lnc-DIF also showed significant effects on rescuing osteoporosis mice (unpublished results), which further proved lncRNAs that carried by certain bone targeting delivery system would serve as a potential therapeutic strategy of osteoporosis in the near future.

6.5

Mechanism Researches of Osteogenic lncRNAs

Our research group had established a simple and efficient method to reveal the mechanisms of lncRNAs. In our study of lncRNA AK016739 and AK045490, RT-PCR assay was made for evaluating the expression levels of the transcription

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factors in multiple essential osteogenic pathways within osteoblasts both treated by si-lncRNAs and overexpression plasmids. We found expression levels of Tcf7 and Lef1, the transcription factors in Wnt signaling pathway were significantly changed by both AK016739 and AK045490. We further adopted luciferase reporter assay with TOPflash system (an essential luciferase reporter plasmid system for Wnt signaling pathway), and the luciferase activities were also affected by this two lncRNAs. So, we confirmed the mechanisms of AK016739 and AK045490 on Wnt signaling pathway. During our study of lncRNA AK039312, AK079370, lnc-DIF, and AK018451, we upgraded our method by correlation analysis of the expression pattern in mice. We have detected the expression level of lncRNA and essential osteogenic transcription factors by RT-PCR in the femur tissue of different ages of C57BL/6 mice. A correlation analysis was made between lncRNA and each osteogenic transcription factors and the result would show the most correlated osteogenic pathway of lncRNA in mice. Taking AK039312 and AK079370 as an example, previous study has found AK039312/AK079370 inhibited osteogenic differentiation, and then the screening revealed the correlation between AK039312/AK079370 and essential osteogenic marker genes and transcript factors. None of the transcript factors that inhibited osteoblast differentiation (Hes1, Smad2, Hif1a) showed positive correlation with AK039312 and AK079370. And for genes and transcript factors that promoted osteoblast differentiation, Bmp4 did not show correlation with AK039312 and AK079370, while Tcf7 and Lef1, downstream transcript factors of Wnt/β-catenin pathway, showed manifestly negative correlation with AK039312 and AK079312 [17]. However, it is still imperfect to determine the targeting signaling pathway using RNA expression levels of transcription factors. The transcription factors function as intra-nucleus proteins, its activities and effects cannot be simply judged by its RNA level. So, our next plan is to construct a NanoLuc luciferase reporter system, the system would contain multiple NanoLuc luciferase reporter plasmids, and within each plasmid, a motif region of one osteogenic transcription factor is inserted. In our further studies, we will use this reporter gene system to screen out potential transcription factors regulated by lncRNAs.

6.6

Osteogenic lncRNA as Endogenous miRNA Sponge

In previous studies, we discovered a series of lncRNAs with high capacity to sequester multiple osteogenic miRNAs, this special lncRNA has been proved to have very strong capacity to regulate osteoblast differentiation and bone formation. A lncRNA that named lnc-DIF was identified as an osteogenic lncRNA through our screening strategy as mentioned above. We first proved its function as an osteogenic differentiation inhibiting factor, thus we named it as lnc-DIF (differentiation inhibiting factor). The analysis of lnc-DIF sequence found 13 sequence repeats in the trailing end part of lnc-DIF. This repeat sequence was 53 nucleotides, and

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slight differences were observed in different repeats. 11 out of the 13 repeat sequences were predicted to bond with miR-489-3p. We claimed the non-repeated region that did not contain repeat sequence as “head” region, while the repeated sequence as “tail” region. Expression plasmids containing lnc-DIF head region (head) and lnc-DIF tail region (tail) were constructed and transfected into MC3T3E1 cells. Lnc-DIF head region, which had no miR-489-3p bonding site, had no effect on miR-489-3p level, while lnc-DIF tail region, which contained repeated bonding sites of miR-489-3p, significantly decreased intracellular miR-489-3p. This bonding effect was further evaluated by constructing luciferase reporter plasmids containing lnc-DIF head region or lnc-DIF tail region. Results also showed that lnc-DIF bonded with miR-489-3p through its tail region and down-regulate miR-489-3p level. To further verify the necessity of the miR-489-3p bonding site for the function of lnc-DIF, expression plasmids with lnc-DIF head and tail region were transfected to MC3T3-E1 cells to investigate its function on osteoblast differentiation. Expression levels of osteogenic marker gene, along with ALP activities and mineralized nodules in lnc-DIF tail region transfected cells were all significantly diminished, while lnc-DIF head region showed no influence. Functions of lnc-DIF head and tail region were also tested in vivo. Bone formation level of lnc-DIF tail region transfected mice was significantly decreased compared to lnc-DIF head region. The results indicated that lnc-DIF tail region inhibited osteoblast differentiation and bone formation through bonding with miR-489-3p (unpublished results). LncRNAs could bond with miRNAs and further sequester them. This sort of lncRNAs were defined as ceRNAs [18, 19]. So far, most lncRNAs that function as ceRNA only have one bonding site for one sort of miRNA, which means one LncRNA can only sequester a single miRNA. This results in low efficiency and low specificity. In our study, we found lnc-DIF contained a special structure of repeat sequences in its trailing end, 11 of 13 repeats were predicted to bond with miR-489-3p which suggested that lnc-DIF may act as an efficient miRNA sponge and one single molecular of lnc-DIF would potentially sequester 11 miR-489-3p. This repeat sequence was proved sequestering multiple miR-489-3p and efficiently inhibited the positive effect of miR-489-3p on osteoblast differentiation and bone formation. The study had proved lnc-DIF as a strong endogenous miRNA sponge, and also provided an important hint that some lncRNAs may also contain endogenous repeats and potentially sequester miRNAs. Manipulating the expression levels of these lncRNAs might be a potential therapy for aging and postmenopausal osteoporosis. Moreover, this would reveal human-derived endogenous miR-4893p sponge or other human-derived endogenous miRNA sponges using the similar method. In our study, two more endogenous miR-489-3p sponges have been detected in human genome, and were also determined as strong regulators of osteoblast differentiation. This method would evade the low homology between different species of lncRNAs and might be developed as potential therapeutic targets of osteoporosis in our further studies.

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Conclusion

LncRNAs have been reported as an important regulator of osteoblast differentiation and bone formation, and has received increasing attention by related researchers. However, due to its complicated mechanisms and low homology between different species, lncRNAs were seldomly considered as a direct therapy for osteoporosis. In our previous studies, we have developed some methods that would overcome the defects on lncRNA researches and reduce the cost of lncRNA researches. We hope these methods would make osteogenic lncRNA study simplified, and thus made osteogenic lncRNAs closer to a therapeutic target for osteoporosis. Acknowledgements The authors would like to thank Yang Yu at the Tianjin Medical University for her generous support and suggestions. This work was supported by the Natural Science Foundation of China (82072106, 31570940, 81772017 and 81801871), the China Postdoctoral Science Foundation (2020M683573, 2018T111099, 2017M610653 and 2017M613210), the New Century Excellent Talents in University (NCET-12-0469), the Shenzhen Science and Technology Project (JCYJ20160229174320053), the Innovation Foundation for Doctor Dissertation of Northwestern Polytechnical University (CX201821).

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Part III

RNA Synthesis Technology and RNA Therapy in Bone Diseases

Chapter 7

Synthetic Technology of Noncoding RNAs Used in Bone Disease Research and Therapeutics Ye Tian, Chong Yin, Chaofei Yang, Mili Ji, Xiaohua Chu, and Airong Qian

Contents 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Viral Vector Mediated RNA Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Chemical Synthesis of RNA Molecules and Their Applications in Bone Disease Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 In Vivo Production of RNA Molecules and Their Applications in Bone Disease Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Noncoding RNAs (ncRNAs) play an essential role in the control of target gene expression underlying various processes of bone cells, thus dysregulation of ncRNAs is involved in the pathogenesis and progression of bone disease. Development of novel RNA-based therapeutics for bone disorders holds the promise to improve the healing of bone. This chapter summarized the technologies used for ncRNA synthesis which were employed in the therapeutic studies of bone-related diseases in the recent 5 years. Viral vector and chemical synthesis approaches were briefly introduced, and the novel biotechnology that was manufacturing recombinant ncRNA agents through microbial fermentation was highlighted. The in vivo fermentation method could achieve an efficient, large-scale, and cost-effective production of recombinant or bioengineered ncRNAs (as much as tens of milligrams from 1 L of bacterial culture), and this approach may shift the paradigm to apply the natural, endogenous-similar biological ncRNA molecules for bone disease research and therapy.

Y. Tian · C. Yin · C. Yang · M. Ji · X. Chu · A. Qian (*) Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China e-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. 2021 A. Qian, Y. Tian (eds.), Noncoding RNAs and Bone, https://doi.org/10.1007/978-981-16-2402-5_7

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Keywords RNA therapy · Bone disease · Synthesis · In vivo fermentation · tRNA scaffold

Abbreviation ADO2 BMSC CDK-MBD CRYAB EIF2AK2 EZH2 FPLC HOST2 HOTAIR IFIT2 IFNB1 LEPR Lnc RNA MALAT1 MAYA MEG3 miRNA/miR NEAT1 PAGE PS R&D ROR ROR1 Rrna SATB2 shRNA siRNA SIRT1 snoRNA SOX4 sRNA TLR tRNA TUG1

Autosomal dominant osteoporosis type 2 Bone marrow-derived mesenchymal stem cells Chronic kidney disease-mineral and bone disorder αB-crystallin Eif2ak2 encoding protein kinase double-stranded RNA-dependent (PKR) Enhancer of zeste homolog 2 Fast protein liquid chromatography Human ovarian cancer-specific transcripts 2 HOX transcript antisense RNA Interferon-induced protein with tetratricopeptide repeats 2 Encoding IFN-β The leptin receptor Long noncoding RNA Metastasis associated lung adenocarcinoma transcript 1 MST1/2-antagonizing for YAP activation Maternally expressed gene 3 microRNA Nuclear-enriched abundant transcript 1 Polyacrylamide gel electrophoresis Phosphorothioate Research and development LncRNA regulator of reprogramming The receptor tyrosine kinase (RTKs)-like orphan receptors Ribosomal RNA Special AT-rich sequence-binding protein 2 Small hairpin RNA Small interference RNA Sirtuin-1 Small nuclear RNA Sex determining region Y-box 4 Small RNA Toll-like receptor Transfer RNA Taurine upregulated gene 1

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Introduction

Emerging evidences have proved the indispensable roles of noncoding RNAs (ncRNAs) in homeostasis of skeleton system [1–4]. And over the last decade, RNA therapy has been proposed as a possible approach to improve or augment the healing of bone [3–6]. Functional RNAs are delivered to bone tissue/cells using viral or non-viral vectors to either sustain or modulate the expression of therapeutic proteins. In vivo and ex vivo strategies have been proposed towards cancer (primary or metastatic), osteoporosis, osteoarthritis, and other bone diseases. RNA therapy has several advantages over competing methods. For example, the targets of ncRNA are extremely abundant, including those undruggable or non-druggable by conventional modes of therapeutics. RNA molecules may permit the re-targeting of mutated or epigenetic changed targets, thus expand the druggable targets from proteins to RNAs as well as the genome [7]. The RNA molecules employed in the therapy of bone diseases are various. A variety of RNA entities such as antisense RNAs, microRNAs (miRNAs), small interfering RNAs (siRNAs), and other forms of small RNAs (sRNAs) are used to treat the diseases through silencing target gene expression [8–10]. The development of novel RNA-based therapeutics is highly challenging due to the unique chemical and pharmacokinetics properties of RNAs compared to conventional small molecules and proteins. Despite the delivery issue of RNA, which is a well-recognized challenge for RNA-based drug R&D, utilization of the proper RNA entities is also critical for the success of RNA therapy. Currently, viral vectors and chemically engineered/synthesized oligonucleotides or RNA “mimics” seem to dominate in RNA research and drug development of bone disorders (Table 7.1). Many viral vectors have been examined for RNA therapies of bone and cartilage, since they are evolutionarily optimized to target and deliver genetic payloads to mammalian cells and tissues [6]. And the chemically synthesized RNA “mimics” are usually decorated with various and extensive chemical modifications to promote their metabolic stability, affinity to targets, or other characteristics on demand [39– 41]. Recently, some researchers start to concern about the differences between the chemical engineered and natural RNAs that are transcribed from the genome [42– 44], because the later carry no or minimal posttranscriptional modifications [45], which is very distinct from chemical produced RNAs. Therefore, large efforts have been made to establish novel biotechnologies for in vivo fermentation production of ncRNAs using bacteria as factories [46–48] since the mammalian and bacteria RNAs share some modifications. Thus, bioengineering of ncRNAs may represent a new type of tools for RNA therapy development to bone diseases. In the following review, we provide an overview on the RNA synthesized technologies which applied to manufacture RNA entities used in bone disease treatment. And we place greatest emphasis on aspects of recent 5-year RNA-based therapy for disorders of the bone. Following a brief introduction of viral vectors and chemical synthesis approaches, we will focus on the novel biotechnology which is the in vivo production of ncRNA molecules in E. coli, because these recombinant or

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Table 7.1 Summary of representative technologies in the past 5 years for ncRNA production used in bone disease treatment Synthesized method Viral vector— lentivirus

Disease/cell Dexamethasone induced injury/ osteoblast Osteosarcoma Osteosarcoma Osteoporosis/hFOB1.19 Osteosarcoma Bone metastasis of breast cancer Estrogen-deficiency osteoporosis/ mesenchymal stem cells Chondrosarcoma

Viral vector— adenovirus Chemical synthesis

Glucocorticoid-induced osteonecrosis of the femoral head/ CD34+ stem cell Bone defects of calvaria Postmenopausal osteoporosis/ BMSC Osteoporosis/hFOB1.19 Osteosarcoma

CDK-MBD/Raw264.7 ADO2 Osteosarcoma

Osteosarcoma Bone metastasis of breast cancer Osteoporosis and rheumatoid arthritis/osteoclast Age-related osteoporosis/endothelial cell Intervertebral disc degeneration

Estrogen-deficiency osteoporosis/ mesenchymal stem cells

Target siRNA of lnc-MALAT

Ref. [11]

shRNA of ROR siRNA of NEAT1 shRNA of SATB2 and lnc MALAT1 shRNA of MNX1-AS1 shRNA of lnc MAYA miR-146a and its antagonist miR-454-3p shRNA of lnc HOTAIR miR-26a

[12] [13] [14]

miR-26a Lnc MEG3

[20] [21]

miR-34c mimic

[14]

siRNA of lnc LUCAT1 miRNA inhibitor of miR-200c Anti-miR-223 Pre-miR-223 siRNA of Clcn7G213R siRNA of lnc TUG1 and SOX4 miR-132-3p mimic and inhibitor siRNA of lnc HOST2 siRNA of lnc MAYA, ROR1 etc. miR-182 mimic and inhibitor siRNA of EIF2AK2 and IFNB1 agomiR-195

[22]

siRNA of SIRT1 miR-141 mimic and inhibitor miR-146a mimic and inhibitor

[29]

[15] [16] [17] [18] [19]

[23] [24] [25]

[26] [16] [27]

[28]

[17] (continued)

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Table 7.1 (continued) Synthesized method

Disease/cell Osteosarcoma

Chondrosarcoma

Metastatic prostate cancer Postmenopausal osteoporosis/ BMSC Chondrosarcoma Osteosarcoma lung metastasis

Overexpression vector pcDNA3.1 pCDH pcDNA3.1(
)

In vivo fermentationhybrid tRNA scaffold

Osteoporosis/BMSC and hFOB1.19 Osteosarcoma Osteosarcoma Chondrosarcoma Estrogen-deficiency osteoporosis/ osteoclast Osteosarcoma lung metastasis Osteosarcoma

Osteosarcoma/pulmonary metastasis Estrogen-deficiency osteoporosis

Target shRNA of IFIT2 miR-645 mimic and inhibitor siRNA of EZH2 and lnc HOTAIR miR-454-3p mimic and inhibitor Pre-miR-145 and anti-miR145 miR-133a-3p mimic and inhibitor miR-181a inhibitor siRNA of CRYAB miR-491 mimic and antisense Lnc MALAT1

Ref. [30]

Lnc TUG1 Lnc LINC00161 Lnc HOTAIR miR-155 and anti-miR-155 siRNA of LEPR miR-491 antisense miR-34a

[25] [30] [18] [34]

miR-34a

[37]

Anti-miR-129

[38]

[18]

[31] [21] [32] [33]

[14]

[33] [35, 36]

Note: the full names of abbreviations used in Table 7.1, please refer to the abbreviation list

bioengineered ncRNAs may better stand for the natural ncRNAs in human body, and could be a new direction for future development of RNA-based drugs in bone healing.

7.2

Viral Vector Mediated RNA Therapy

Several types of viruses have been modified in the laboratory for use in gene therapy long time ago [49, 50]. Particular viruses have been selected as gene/RNA delivery vehicles because they are highly efficient and long-lasting at nucleic acid delivery to

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cells [51–53]. Now, it is a well-established technical for RNA therapy. For example, lentiviruses or adeno-associated viruses can be applied to deliver either pre-miRNAs to increase their levels or, on the contrary, the antisense miRNAs to decrease their levels. Many researchers have employed the viral vectors to intracellularly express ncRNAs, including small hairpin RNAs (shRNA), siRNAs, miRNAs and their agonists, to treat various bone diseases (Table 7.1). The targets of these ncRNAs could be long noncoding RNAs (lnc RNAs), mRNAs, and miRNAs (Table 7.1). And lentiviral vectors seem more popular than other viral vectors (Table 7.1), may be due to they can safely transduce bone tissues and provide sustained levels of gene expression [54]. Since the viral expression systems are DNA-based, the real expression levels or doses of ncRNA products are greatly variable and hard to determine. In addition, the expression of ncRNA products does not strictly correlate with the amount of DNA plasmid administered to the cells. Because both the transfection/infection efficiency and the biogenesis machineries of RNA in host cells will influence the generation of target ncRNA products. Besides, potential insertional mutagenesis, accessory protein sequences in the packaging constructs, immune response to viral proteins, etc. are all possible disadvantages for viral systems [51, 54]. Except viral vectors, some non-viral plasmids are also constructed and utilized to over express ncRNAs in cells/ tissues (Table 7.1).

7.3

Chemical Synthesis of RNA Molecules and Their Applications in Bone Disease Therapy

Standard phosphoramidite chemistry has been automated and widely used for the manufacture of oligonucleotides including RNAs [55]. Many kinds of ncRNAs, such as anti-miRs or mimics can be synthesized chemically with various artificial modifications. The chemical modifications can improve the diversity of RNAs and enhance the stability and/or increase binding affinity of synthetic RNA molecules [39–41, 56]. Commonly, O-methyl (OMe), fluoro (F), O-methoxy-ethyl (MOE), and locked nucleic acid (LNA) are used to mask or substitute the so-called vulnerable 20 -hydroxy group of ribose, and phosphorothioate (PS) is subjected to change the phosphate backbone (Fig. 7.1). The chemical modifications of synthetic RNA molecules have been comprehensively reviewed by many researchers [40, 42, 43, 56]. Chemical synthesized RNA agents are easily obtained through commercial vendors, such as Thermo Fisher Scientific Inc., Shanghai GenePharma Co., Ltd., and Guangzhou RiboBio Co., Ltd. And these agents are widely applied for research and development of new therapeutic strategies of bone-related diseases (Table 7.1). Kazuki et al. used LNA oligonucleotides specifically targeting gene Eifrak2 to mechanistically identify PKR (protein kinase double-stranded RNA-dependent) as

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Fig. 7.1 Common chemical modifications of RNA analogs

a new and essential target of miR-182, then finally revealed the network mediated by miR-182-PKR-INF (interferon)-β axis in osteoclastogenesis and the therapeutic implications of miR-182 suppression in osteoprotection [27]. LNAs are nucleic acid analogs that are introduced to form a bridge between the 20 oxygen and 40 carbonyl in the furanose ring. This locking effect restricts the conformation of ribose in A-form position to enhance base stacking interactions, which forms the basis for the enhanced hybridization properties [57, 58]. The mirVana™ miRNA products provided by Thermo Fisher Scientific Co. (USA) are also commonly used in research on prevention and treatment of skeletal system diseases. MiR-141 (mirVana™ miRNA mimics/inhibitors) was utilized to investigate its key role in the intervertebral disc degeneration (IDD) through in vitro and in vivo experiments, and the blockage of miR-141 in vivo might accomplish as a potential therapeutic approach in the treatment of IDD [29]. MirVana™ miRNA mimics/inhibitors are small, double/single-stranded RNAs that mimic/inhibit endogenous mature miRNAs and enable miRNA functional analysis by up/down-regulation of miRNA activity, respectively. The chemical modification is included in mirVana™ miRNA agents to enhance specificity and lower off-target effects. PS-modified RNA agents are also introduced to bone disease treatment. MiR-182 inhibitor with fully PS-modified backbones to enhance stability, pharmacokinetic and pharmacodynamic properties in vivo tested its bone protection effect in osteoporosis and rheumatoid arthritis mouse models [27]. From the above cases we can see chemical modified RNA

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agents remain as major tools for ncRNA research and RNA-based drug development in bone disease therapy. Although chemical modification can add some benefits to synthetic RNA molecules, such as enhanced stability and pharmacokinetic properties (e.g., longer half-life) [59, 60], there are still some disadvantages existed. Firstly, it has been speculated that nucleoside modifications may trigger the host immune system [61]. The artificial “decoration” makes the synthetic RNA molecules totally different from natural ncRNAs, thus they may be discriminate as pathogenic RNA, on the contrary, naturally occurring nucleoside modification lacks immune-activating properties [61–63]. It has been revealed that RNA molecules with subtle differences in sequence and structure activate distinct families of toll-like receptors (TLRs) [64, 65], which hints at the importance of RNA modifications. Therefore, the modification of synthetic RNA molecules needs to be carefully selected to avoid stimulate innate immune system considering the future clinical applications. Unfortunately, all the research works listed in Table 7.1 that used chemical synthesized RNA agents failed to test the immune response of the animals who received the dosage of ncRNAs. In addition, although chemical synthesis of RNAs has been automated, chemo-engineered ncRNA agents remain expensive, especially the cost of synthetic RNA increases quickly with a parallel increase in RNA length and number of modifications.

7.4

In Vivo Production of RNA Molecules and Their Applications in Bone Disease Therapy

Great efforts have been made to develop new biotechnologies for the in vivo manufacture of ncRNA molecules in awareness of the shortages of chemically synthesized RNA agents, e.g., the modification at the ribose could influence the global structure of RNAs that govern their stability, plasticity, interactions with partners or ligands, because the ribose and the unconjugated 20 -hydroxyl group are key elements for the elaborate structure, biological function, and safety profile of natural RNA molecules [66]. The bioengineered ncRNA molecules are believed to carry natural modification and fold properly as “true” endogenous ncRNAs, thus could have minimal immune-activating properties [43, 44, 67]. Besides, manufacturing ncRNA agents in living cells is able to cut down the cost of experiment, because synthetic ncRNAs are usually provided in micromolar scale, which is far from milligram quantities needs for animal studies and clinical research, while in vivo fermentation approaches are expected to provide large quantities of ncRNA agents [42, 43, 67]. Many strategies have been adopted to develop new fermentation-based method to cost-effectively bio-synthesize ready-to-use ncRNA agents with biological

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functions. To avoid the degradation of RNases within the host cells, the target ncRNAs should be presented as stable molecules, protected by a stable complex or “masked” as endogenous RNA entities. For example, Huang et al. used p19, a plant viral siRNA-binding protein, to stabilize the bacterial siRNAs, their optimized method could achieve a yield of ~10 mg purified siRNA per litter bacterial culture with high efficiency of gene silencing [46, 68, 69]. Some stable RNA scaffolds or carriers are also employed to generate various types of target ncRNAs (e.g., miRNAs, siRNAs, and RNA aptamers). The ncRNAs of interest can be fused into these scaffolds resulting chimeric constructs to accumulate in the cytoplasm to a sufficiently high level. The 5S rRNA (ribosomal RNA) of E. coli has been exploited to serve as a scaffold for the production of recombinant ncRNA species [70, 71]. Also, tRNA (transfer RNA) and hybrid tRNA/pre-miRNA can work as stable ncRNA carriers [44, 48]. These genetically engineered recombinant RNAs may represent a new class of biological RNA agents for research and development. In the field of bone disease research, it has been started to explore the usage of bioengineered ncRNA agents for development of RNA therapy. Zhao et al. generated recombinant pre-miR-34a in E. coli using tRNA scaffold, and then tested its suppression effect towards osteosarcoma in vitro and in vivo [35]. Besides, the blood chemistry profiles in their study exhibited well tolerance by animals under their therapeutic doses [35]. Moreover, they quested the synergistic suppression of osteosarcoma by the combination therapy of recombinant miR-34a and chemotherapy drug doxorubicin [36]. Then Jian et al. further found the optimal effects for tripledrug combination in osteosarcoma and pulmonary metastasis treatment with bioengineered miR-34a, doxorubicin, and sorafenib targeting RNA translation, DNA replication, and protein kinase signaling, respectively [37]. With the development of fermentation-based technology, it is possible that more and more researches will adopt bioengineered recombinant ncRNAs to treat bone diseases. The in vivo production of large quantities of RNA molecules using tRNA as a scaffold was firstly reported in 2007, motivated by the idea to generically express and purify structured RNA using tools that parallel those available for recombinant protein [72]. Choosing tRNA as scaffold, because it is the simplest stable RNA in cells, and its three-dimensional four-leaf clover structure enables resistance to heatdenaturation and nuclease cleavage [73]. Methionine-tRNA (tRNAmet) with target RNA infused at its anticodon region was overexpressed in E. coli using a strong lipoprotein (lpp) gene promoter and a ribosomal RNA operon transcription terminator (rrnC) [48]. The acceptor, TψC and D stems are retained to maintain the tertiary structure of tRNA and helped the recombinant RNA chimera presumably recognized by the cells as their own tRNA species to escape RNase degradation and highly accumulate in E. coli [48, 74, 75]. The tRNA scaffold has been used for the production of various target RNAs successfully, including pre-miRNAs [35, 36, 48, 72, 75–82], aptamers and viral RNAs [48, 72], snoRNAs (small nuclear RNA) [83], hammerhead riboswitch RNAs [75] as well as co-expression of RNA-protein complexes [84]. And some researches demonstrated that recombinant RNA that fused

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with tRNA scaffold showed minimal immune response in mouse which indicated by IL-6, the most sensitive pro- and anti-inflammatory cytokine in response to nucleic acids [78]. Although the tRNA scaffold approach shows great promise, the success and efficacy in heterogenous expression of chimeric RNAs largely depend upon the structure of target RNAs and resistance to bacterial RNase, which makes this approach seems less robust. The hybrid tRNA/pre-miRNA molecules have been established recently on the basis of tRNA scaffold as a novel ncRNA carrier [47, 85]. The miRNA (e.g., miR-34a-5p) duplexes within the tRNA/pre-miRNA (e.g., tRNAMet/pre-miR-34a) scaffold can be replaced directly by miRNA, siRNA, or sRNA (small RNA) of interest, along with its complementary sequence. A wide variety of RNAs have been generated through hybrid tRNA/pre-miRNA carrier [47, 85–91]. And when the recombinant ncRNAs cross the cellular barrier, they can be processed to their mature form through the endogenous miRNA biogenesis machinery. Yin et al. investigated the rescue effect of recombinant miR-129-5p to menopause osteoporosis [38]. They produced the miR-129-5p inhibitor by hybrid tRNAMet/pre-miR-34a scaffold in E. coli, and found the bioengineered inhibitor promoted osteoblast differentiation and greatly ameliorated menopause osteoporosis [38]. It was reported that the hybrid tRNAMet/pre-miR-34a was able to accumulate in bacteria to a very high level (e.g., 10–20% of total RNAs) [47, 78], which solved the problem that with direct heterogenous expression in bacteria, human pre-miRNAs did not accumulate to an adequate level for purification, and there were big variations in RNA expression and a low overall success rate of high-yield even when using tRNA scaffold [47]. Optimization of hybrid tRNAMet/pre-miR-34a scaffold by changing pre-miR34a sequence G138U/139ΔG can further improve the expression levels of ncRNAs (e.g., >30% of total bacterial RNA) and success rate (~80%) for the manufacturing of target RNAs [85]. Moreover, tRNA/pre-miR carrier can accommodate other pre-miRNAs (or shRNA with stem-loop structure) to co-express multiple small RNAs simultaneously [92]. And the immunogenicity seems low for ncRNAs produced through hybrid tRNA/pre-miR scaffold [89], which makes it very promising for clinical application. The overall technical process for bioengineering ncRNA molecules with hybrid tRNA/pre-miR scaffold is as follows (Fig. 7.2). The coding sequence of target ncRNA is cloned into a derivative of pBSMRNA vector. After transformation and fermentation, E. coli (e.g., HST08) is collected. The expression of target ncRNA is detected by RNA gel electrophoresis, a band appearing between 150 and 300 nt indicates the accumulation of target ncRNA. Spin column and FPLC (fast protein liquid chromatography) can be employed to purify target ncRNA in small- and large scale, respectively. The purity of final ncRNA product can be measured by HPLC (high performance liquid chromatography).

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Fig. 7.2 Bioengineering ncRNA molecules for research using hybrid tRNA/pre-miR scaffold. The ncRNA coding sequence is inserted into a target vector through restriction sites and transformed in E. coli. After fermentation overnight, bacteria are harvested and total RNAs are extracted. The overexpression of target ncRNA is verified by Urea-PAGE. Recombinant RNAs are then purified by anion exchange FPLC method. Pure recombinant RNAs are subjected to in vitro and in vivo investigation of biological activity, effectiveness, and safety properties, etc

7.5

Conclusion

Over the past few years, remarkable progress has been made in defining ncRNAs’ functions in bone formation, remodeling, fracture repair, and other bone-related diseases. Emerging evidences have pointed out that ncRNAs play a multi-faceted

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role in bone disease, which undoubtedly a new breakthrough in the field of bone healing. RNA-based therapy strategy has been developed to treat bone disease in many preclinical researches, while the road to clinical evaluation requires more efforts. One of the challenging is to carefully select the RNA entities to assure the success of clinical application. Till now, the techniques for ncRNA production which are used in bone disease treatment include viral vectors, chemical synthesis, and in vivo fermentation. Although the first two techniques dominate in RNA research and drug development of bone disorders currently, there are still some concerns need to be thought over. For example, it is hard to accurately quantity the ncRNA levels generated form viral vectors, and the artificial modifications introduced in chemically synthetic RNA molecules make them very different from natural endogenous RNAs. And the triggering of immune response also demands consideration when applying these two techniques, which are missing in most of the current preclinical researches. In contrast, the bioengineered ncRNAs manufactured by in vivo fermentation, carry no or minimal posttranscriptional modifications which is like natural RNAs, are biologically active in regulating target gene expression and cellular processes, and are well tolerated in animal models. Among them, the hybrid tRNA/pre-miRNA scaffold-based technology has shown to be a more robust and versatile platform to cost-effectively produce various biologic RNA agents in largescale including those related to bone disorders. Though there are not many studies that involved bioengineered ncRNAs in orthopedics now, future research shall explore novel strategies and critically assess the utilities of recombinant ncRNAs in bone disease therapy. Acknowledgements This work was supported by the Natural Science Foundation of China (82072106, 32000924, and 81801871), the China Postdoctoral Science Foundation (2020 M683573, 2019 T120947, and 2017 M613210), Natural Science Basic Research Plan in Shaanxi Province of China (2018JQ8032 and 2018JQ3049), Shaanxi Provincial Key R&D Program (2018KWZ-10), Shaanxi Postdoctoral Science Foundation (2017BSHEDZZ13), Special Fund for Technological Innovation of Shaanxi Province (No. 2019QYPY-207), the Fundamental Research Funds for the Central Universities (3102018zy053).

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Chapter 8

RNA Therapy in Bone Diseases Jiawei Pei, Qian Huang, Mili Ji, Xiaohua Chu, Ye Tian, Airong Qian, and Peihong Su

Contents 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 The Therapeutics of ncRNA to Osteoarthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 The Therapeutics of ncRNAs to Osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.1 The Therapy of miRNA to Osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2 The Therapy of LncRNA and siRNA to Osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4 The Therapeutics of ncRNA to Bone Tumor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.1 The Therapy of ncRNA to Primary Bone Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.2 The Therapy of ncRNA to Metastatic Bone Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Bones are constantly modeling and remodeling in a stable internal environment. When the homeostasis is damaged with age growth and pathological conditions, bone diseases, such as osteoarthritis (OA), osteoporosis (OP), bone tumor, and so on, will occur. Bone diseases represented severe public health threats with limited therapy approach. However, traditional medicine treatment for these bone disorders had certain limitations. Increasing evidences revealed that diverse ncRNAs play critical roles in occurrence of the bone disorders. In the recently decades, more and more studies prompted that targeting ncRNAs might become an attractive and new therapeutic strategy for bone related diseases. This chapter will

J. Pei · Q. Huang · M. Ji · X. Chu · Y. Tian · A. Qian Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China e-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected] P. Su (*) Institute of Basic and Translational Medicine, Xi’an Medical University, Xi’an, Shaanxi, China e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2021 A. Qian, Y. Tian (eds.), Noncoding RNAs and Bone, https://doi.org/10.1007/978-981-16-2402-5_8

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highlight the recent advance in the development of ncRNA in bone disease and discuss the possibilities for RNA therapy in bone disease treatment. Keywords ncRNA · Bone diseases · RNA therapy · Recent advance · Treatment

Abbreviation CS Ctgf DEPTOR DNMT1 ER ES ESFT HMGB1 IHH ITGA6 LncRNA Lrp5 MIA MiRNA MK2 MO MSCs NcRNA NF-kB NP OA OP OS PCA PR PU SiRNA SPHK1 Wnt

8.1

Chondrosarcoma Connective tissue growth factor DEP domain containing m-TOR interacting protein DNA methyltransferase1 Estrogen receptor Ewing’s sarcoma Ewing sarcoma family tumor High mobility group box 1 Indian hedgehog Integrin Alpha 6 Long noncoding RNA Low-density lipoprotein receptor-related proteins-5 Mono-iodoacetate-induced arthritis MicroRNA Mitogen-activated protein kinase-activated protein kinase 2 Multiple osteochondromatosis Mesenchymal stem cells Noncoding RNA Nuclear factor kappa-B Nanoparticle Osteoarthritis Osteoporosis Osteosarcoma Prostate cancer Progesterone receptor Polyurethane Small interfering RNA Sphingosine kinase-1 Wingless/integrated

Introduction

Bones are constantly modeling and remodeling in a stable internal environment. When the homeostasis is damaged with age growth and pathological conditions, bone diseases, such as osteoarthritis (OA), osteoporosis (OP), bone tumor, and so on,

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will occur. Nowadays, these bone diseases represent severe public health threats with limited therapy approach [1]. At present, the treatment for bone diseases is mainly surgery and drug therapy. Traditional drugs usually interact with certain proteins that are common targets of current pharmaceutical drugs for disease control. However, the proteins structure could be altered by genetic or epigenetic changes, which induced escape from current medication [2]. RNA therapy is a novel approach to treat bone related diseases by regulating the gene expression through binding with target RNA or DNA sequence [3]. More and more studies revealed that targeting ncRNAs might become an attractive and new therapeutic approach for bone related disease [4]. RNA molecules are a new class of therapeutics that may interact with mutated targets, which present great promise to expand the range of druggable targets from proteins to RNAs as well as the genome. RNA therapy is a novel approach to treat bone diseases by regulating gene expression through ncRNA, such as siRNA or miRNA, etc. [3]. More and more studies revealed that targeting ncRNAs might become an attractive and new therapeutic approach for bone related diseases [4]. This chapter will highlight the recent advances in the development of ncRNA treatment for osteoarthritis (OA), osteoporosis (OP), bone tumor and explore the possibilities for applying RNA therapy in bone disease treatment.

8.2

The Therapeutics of ncRNA to Osteoarthritis

More and more evidences showed ncRNAs play important roles in pathogenesis of bone diseases [5–7]. Hence, ncRNAs become important therapeutic targets for bone diseases due to advantages such as overcoming chemotherapy resistance, high efficiency, and low toxicity. OA is a complex multigenic disease. Over the past decades, although a growing number of pathways are implied in OA pathogenesis including Wnt signaling, NF-kB, apoptosis, autophagy, cell cycling, Notch, and others [8, 9], there is limited option for OA treatment. Compared with other ncRNAs, siRNA, miRNA show huge potential and abilities to target “non-druggable” targets, which could be designed to act on virtually any genes [10]. Table 8.1 lists some potential therapeutic targets related to OA. There was no ongoing clinical trials for use of ncRNA in OA treatment according to clinical trial gov although many ncRNA candidates were approved in clinical trials to treat other diseases [17]. One of the challenges of ncRNA treatment for OA is the lacking of suitable delivery vehicle to carry ncRNA to OA region efficiently and safely. Numerous studies have used intra-articular injection of microRNA- or inhibitorexpressed adenovirus or lentivirus in mouse and rat models of OA. Dai et al. injected miR-101 inhibitor loaded by adenovirus vector into mono-iodoacetate-induced arthritis (MIA) rat’s knee joint and found that miR-101 inhibitor could prevent cartilage degradation after 14 days detecting [18]. Wang et al. found lentivirus

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Table 8.1 Some potential therapeutic targets related to OA ncRNA MiR-377-3p

Target gene ITGA6

MiR-140-5p

HMGB1

MiR-103

SPHK1

MiR-455-3p

PAK2

Si-Dickkopf-3

Dickkopf-3

Si-MK2

MK2

Effects Alleviating IL-1β-caused chondrocyte apoptosis and cartilage degradation Affects chondrocyte proliferation, apoptosis, and inflammation Promoting chondrocyte apoptosis by down-regulation of sphingosine kinase-1 and ameliorates PI3K/AKT pathway in osteoarthritis Promoting TGF-β signaling and inhibits osteoarthritis development by directly targeting PAK2 Protecting cartilage integrity by preventing proteoglycan loss and restore OA-relevant signaling pathway activity Mediating the downstream effects of p38 activation on PGE2 release and the expression and release of catabolic proteases

Ref. [11] [12] [13]

[14] [15] [16]

mediated-antago-miR-483-5p injection delayed the progression of experimental OA [19]. The challenge for viral vectors is biological tissue safety, including possible carcinogenicity, viral toxicity, immunogenicity, etc. [20]. Therefore, compared with viral vector, non-viral vector is emerging as a promising approach for delivery of siRNA or miRNA in vivo with lower toxicity, cost, and immunogenicity [21]. Wang et al. injected siRNA of Indian hedgehog (Ihh) delivered by a novel nanoparticle into mice knee joint and revealed the siRNA injection attenuated cartilage degeneration [22]. However, although non-viral vectors can deliver siRNA or miRNA efficiently and safely to some extent, delivery efficiency and safety remain a huge challenge because of immune response, pharmacokinetics, toxicity issues, and so on. Effects have been put into researching non-viral suitable delivery systems, but few vectors are ongoing in clinical trial. In recent years, the efficacy of mesenchymal stem cells (MSCs) in cartilage repair has been demonstrated in animals [23, 24] and clinical trials [25, 26]. MSC derived exosomes therapies were effective in cartilage repair in animal studies [27]. Tao et al. injected MSC derived exosomes containing miR-1405p into knee joint, and found cartilage tissue regeneration was enhanced in rat [28]. Exosomes can avoid the RNA drugs being degraded and can circulate in vivo for a long time. Therefore, the ncRNA-containing exosomes derived from modified cells hold potential as future therapeutic strategies. Other kinds of nanoparticle complexes were developed and were used to deliver RNA molecules [29, 30]. Yan et al. developed a self-assembling peptide nanoparticle (NP) with a cell penetrating peptide and was complexed to siRNA of NF-κB p65. They found the peptide-siRNA NP freely and deeply penetrated into all layers of human cartilage. The siRNA was detected in persisted up to 3 weeks [31]. Despite the lack of animal models, it was demonstrated that NP could be a potential drug delivery vehicle to treat OA.

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Currently, most common delivery methods are local knee joint injection with the advantages that are high local administration concentration, long drug duration, good treatment effect, and low systemic side effects. Systemic administration is another treatment method for OA [32]. This method needs large amounts of RNA, cost expensive, high requirements for protecting RNA from degradation. After many years researching, the pathogenesis of OA remains incompletely understood and most of the pathogenesis of osteoarthritis come from surgical posttraumatic diseases. Because of dense extracellular matrix of cartilage renders the chondrocytes inaccessible, even to intra-articular injections, there are few specific drug for OA treatment [33]. Although the pathogenesis of OA is complex and no RNA drugs under clinical, many RNA molecules targeting candidate genes of OA were tried in scientific studies (Table 8.2). Since RNA therapy has a wide range of targets, it can regulate the expression of genes in the early stage of the disease and affect the expression of related pathogenic proteins. In theory, according to the sequence of the disease-causing protein, corresponding ncRNA can be designed as disease intervention. However, the challenge for RNA therapy is the delivery system and sequence optimization. With the development of technology, a variety of delivery systems have been applied in preclinical and clinical research [3, 45, 46]. The safety and efficacy of RNA drugs have always been concerned. Hence, the optimization of delivery vector and RNA sequence modification is a problem that RNA therapy needs to face in the future.

8.3

The Therapeutics of ncRNAs to Osteoporosis

The current pharmaceutical treatment strategies for osteoporosis contain bisphosphonates, selective estrogen receptor modulator, calcitonin, teriparatide, etc. However, side effects of these drugs limit their long-term applications. In the recent several decades, ncRNAs attracted more and more attention in osteoporosis therapy due to their specific features.

8.3.1

The Therapy of miRNA to Osteoporosis

When it comes to miRNA therapy, local injection of miRNA is the easiest and most direct way. Gu et al. found miR-497 promoted osteoblast proliferation and collagen synthesis through TGF-β1/Smads signaling pathway. MiR-497 mimics injection through hip joints reduced bone marrow cavity and elevated trabeculae with intact structure and neat arrangement in bone tissue of OVX rat, which suggested miR-497 had therapeutic effect on rat’s osteoporosis [47]. Similarly, Chen et al. found miR-503 antagomir (special chemically modified miRNA agonists) injection prevents bone loss by inhibiting bone resorption in vivo when they injected miR-503

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Table 8.2 The therapy of ncRNA to OA Drugs MiR-101 inhibitor

Formula Adenovirus

Administration Knee joint

Period 2 weeks

Antago-miR-4835p MiR-128a antisense oligonucleotide MiR-140 antagomir Indian hedgehog siRNA

Lentivirus

Knee joint

5 weeks

Lentivirus

Knee joint

8 weeks

Chemical modification Lipid nanoparticle

Knee joint

12 weeks

Knee joint

10 weeks

Thyroid hormone receptor alpha (THRα) siRNA MiR-7 mimics

Chemical modification

Knee joint

8 weeks

Lentivirus

Knee joint

6 weeks

IκBζ-siRNA

Adenovirus

Knee joint

8 weeks

IL-β siRNA TNF-α siRNA

Lentivirus

Knee joint

4 weeks

HIF-2α-siRNA

Nanoparticles

Knee joint

7 weeks

p66hsc-siRNA

Nanoparticles

Knee joint

4 weeks

LncRNA LOC101928134 siRNA

Adenovirus

Tail vein

4 weeks

Matrix Metalloproteinase13 (MMP-13) siRNA MiR-210 mimic

Chemical modification

Knee joint

8 weeks

Lentivirus

Knee joint

20 days

MiR-92a-3p

Exosomes

Knee joint

4 weeks

MiR-140-5p

Exosomes

Knee joint

3 weeks

Efficacy Reduced cartilage degradation Delay development of OA Repress chondrocyte autophagy and delay OA progression Alleviate OA progression Attenuate the pathological progress of OA Ameliorated cartilage degradation Attenuates OA progression Attenuates development and progression of OA Lessen the degree of damage to the joints and alleviate joint degradation Prevent cartilage in arthritic mice Decrease cartilage damage Suppress cartilage cell apoptosis of cartilage cell, reduce knee joint cartilage damage of OA rats Delayed cartilage degradation at the early stage of OA development Decreased inflammation in articular cavity in OA rats Inhibit early OA progression and prevent the severe damage to knee cartilage in OA mice model Delayed and the knee joint cartilage damage

Ref. [18] [19] [34]

[35] [22]

[36]

[37] [38]

[39]

[40] [41] [32]

[42]

[43]

[44]

[28]

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antagomir wrapped by lentivirus into OVX mice through tail vein [48]. Moreover, silencing miR-106b accelerated osteogenesis of MSCs and rescued osteoporosis after lentivirus-mediated miR-106b inhibitor was injected [49]. Li et al. revealed similar phenomenon by implanting baculovirus-engineered BMSCs that expressed miR-214 sponge. They found that allotransplantation of the miR-214 spongesexpressing BMSCs healed the defect and ameliorated the bone quality at 4 weeks post-implantation [50]. MiR-138 is a negative regulator of bone formation and silencing of miR-138 enhanced ectopic bone formation in vivo after hMSCs transfected with anti-miR-138 were implanted [51]. Exosomes miRNA is an alternative candidate for miRNA therapy of osteoporosis. Qiu et al. revealed that local injection of BMSCs derived exosomes containing miR-150-3p upregulated Runx2 and Osterix expression, and promoted bone status markers in femoral tissues of OVX mice [52] (Table 8.3). Target delivery systems of miRNA have greatly improved the efficiency of miRNA therapy. Wang et al. have reported that miR-214 antagomir (miRNA antagonist with special chemical modification) loaded by delivery systems target to bone formation surface had therapeutic effect to bone formation of OVX and HLU mice models [55]. Intra-bone marrow injection of antagomir-188 loaded by an aptamer delivery system increased bone formation and decreased bone marrow fat accumulation in aged mice [56]. Anti-miR-214 was delivered by a novel nanoparticle deliver system-Asp8 polyurethane (PU) nanomicelles to osteoclasts, which improved bone microarchitecture and bone mass in OVX mice [57] (Table 8.3).

8.3.2

The Therapy of LncRNA and siRNA to Osteoporosis

At the recent decade, more and more researchers attempted to reveal mechanism and therapeutic strategies of osteoporosis by focusing on the relationship between occurrences of osteoporosis and lncRNA, and the therapy of lncRNA to osteoporosis. A recent study reported by Wang et al. showed that lncRNA OGRU overexpression promoted osteoblasts function after OGRU was delivered to bone formation surface by (DSS) 6-liposome delivery system [59]. Similar efficacy was observed when lncRNA ODSM was delivered into HLU mice by (Asp-Ser-Ser) 6-liposome by Wang et al. [60]. However, the most common method for osteoporosis treatment is by silencing lncRNA employing siRNA. Li et al. reported in 2018 that local intramedullary injection of DNA methyltransferase1 (DNMT1) siRNA (siDNMT1) attenuated histopathological changes and bone microstructure declines in rat with early disuse osteoporosis [61]. Another study showed that subcutaneous transplantation of hBMSCs containing shRNA of DEP domain promoted BMSC osteogenesis in vivo [62]. Wang et al. found that intraperitoneal injection of osteoclasts transfected with siRNA of LINC00311 inhibited osteoblast proliferation and differentiation and partially rescued osteoporosis in OVX rat [63]. Besides, exosomes containing lncRNA have been used to treat osteoporosis through transplantation of exosomes as well. Yang et al. observed that BMSCs derived exosomal

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Table 8.3 The therapy of miRNA to osteoporosis Drugs MiR497mimics

Formula –

Administration Local injection

Period 2 weeks

MiR-34c antagomir

–

2/week

MiR-503 antagomir MiR-106b inhibitor

Wrapped by lentivirus Wrapped by lentivirus

Bone marrow cavity injection Tail vein injection Local injection

MiR-214

Baculovirusengineered BMSCs that expressed miR-214 sponge HMSCs transfected with antimiR-138 BMSCs derived exosomes BMSCs derived extracellular vesicleencapsulated Loaded by delivery systems target to bone formation surface Loaded by an aptamer delivery system

AntimiR138 MiR-1503p MiR-223p MiR-214 antagomir

MiR-188 antagomir

Anti-miR214 MiR-34a mimics

Loaded by nanoparticle deliver systemAsp8 polyurethane Loaded by a chitosan (CH) nanoparticle vehicle

3 weeks 2 weeks

Bone marrow implant

4 weeks

Ectopic implanted Local injection

4 weeks

Bone marrow cavity injection Tail vein injection

3 weeks 2 weeks

4 weeks

Intra-bone marrow injection

4 weeks

Tail vein injection

1/week

Intravenous injections

2/week for 4– 5 weeks

Efficacy Reduced bone marrow cavity, elevated trabeculae Alleviated the symptoms of osteoporosis Prevent bone loss, inhibit bone resorption Accelerated osteogenesis, rescued osteoporosis Healed the defect, ameliorated the bone quality Enhanced ectopic bone formation Promoted bone status markers Promoted BMSC osteogenic differentiation Promoted bone formation

Increased bone formation, decreased bone marrow fat accumulation Improved bone microarchitecture and bone mass Attenuated postmenopausal osteoporosis

Ref. [47]

[53]

[48] [49]

[50]

[51] [52] [54]

[55]

[56]

[57]

[58]

lncRNA MALAT1 injection into bone marrow cavity alleviated the symptoms of osteoporosis in OVX mice [53] (Table 8.4). These studies implied that lncRNA will be a potential candidate target for osteoporosis therapy in the future. The treatment of osteoporosis using combination of ncRNAs was also studied by researchers recently. Li et al. found that the combination of si-LNC_000052 and agomir-miR-96-5p had better therapeutic effects to postmenopausal osteoporosis. Because when they injected BMSCs co-transfected with si-LNC_000052 and agomir-miR-96-5p into the rats via the caudal vein, symptoms of osteoporosis

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Table 8.4 The therapy of lncRNA and siRNA to osteoporosis Drugs LncRNA OGRU LncRNA ODSM

Formula Loaded by (DSS) 6– liposome Loaded by (asp-Ser-Ser) 6-liposome

Administration Intravenous injections

Period 3 consecutive injections

Efficacy Promoted osteoblasts function

Ref. [59]

Local injection

3 consecutive injections

Attenuated bone architecture and mechanical properties Attenuated bone microstructure declines Promoted BMSC osteogenesis

[60]

Si-DNMT1

–

Intramedullary injection

4 weeks

SiDEPTOR

HMSCS infected by sh-DEPTOR Osteoclast transfected with si-LINC0031 Exosomes

Transplanted subcutaneously

8 weeks

Transplanted subcutaneously

3 months

Inhibited osteoblast variety, rescued osteoporosis

[63]

Periosteal injection Local injection

2/week

Alleviated the symptoms of osteoporosis Alleviated unloading-induced bone loss Rescue bone formation

[53]

SiLINC00311

SiMALAT1 Si-HCG18

SiAK016739

Wrapped by lentivirus –

Subcutaneous injection

Every day for 3 consecutive days 2/day for 10 days

[61]

[62]

[64]

[65]

Table 8.5 The combination therapy of ncRNA to osteoporosis Drugs Si-LNC_000052 and agomir-miR96-5p SiRNA of lncAK039312 and lncAK079370

Formula BMSC co-transfected with ncRNA –

Administration Tail vein injection

Period 3 months

Subcutaneous injection

11 days

Efficacy Improve the therapeutic effects of BMSCs Rescued bone formation

Ref. [66]

[67]

were better alleviated [66]. Yin et al. got similar results when they combined siRNA of lncAK039312 and lncAK079370 to treat OVX mice [67] (Table 8.5). In summary, osteoporosis treatment has been studied by several decades and ncRNAs would be promising candidates for osteoporosis treatment. Treatment efficiency of osteoporosis will be greatly improved by proper delivery system, e.g., nanoparticle and cell engineering method. Meanwhile, combination of different kinds of ncRNAs may have better efficacy than single ncRNA alone.

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The Therapeutics of ncRNA to Bone Tumor

Bone tumors occur in bones or their accessory tissues. Its main characteristics are rapid development, easy metastasis, poor prognosis, and high mortality. At present, the management of bone tumor is based primarily on neo-adjuvant and adjuvant chemotherapy and surgical resection. Radiotherapy is not effective as some bone tumors are relatively radioresistant. And the optimum management of these patients has not been standardized yet due to several patterns of metastatic disease harboring different prognosis. Therefore, it is urgent to develop new treatment methods. The appearance of ncRNA may provide new ideas for the treatment of bone tumors.

8.4.1

The Therapy of ncRNA to Primary Bone Tumors

Primary bone tumors refer to a class of tumors derived from bone tissue, which mainly include osteosarcoma (OS), chondrosarcoma (CS), and Ewing’s sarcoma (ES) etc. At present, studies have confirmed that ncRNA expressing disorders are related to the pathogenesis and progress of many cancers including OS, CS, ES, etc. Yuan et al. injected miR-20a loaded by lentivirus vector into the flanks of nude mice. The data revealed that the miR-20a overexpression could inhibit tumor growth in vivo [68]. Zhang et al. found that BALB/c nude mice were implanted subcutaneously with stably expressing SaOS-2/LV-miR-134, which significantly inhibited the growth of implanted tumors and reduced the tumor weights [69]. At the same time, lung metastases of OS are an urgent matter for survival rate affection of patients. Wang et al. revealed that miR-193a-3p agomir local injection inhibited xenograft tumor growth and reduced doxorubicin resistance in nude mice [70]. Meanwhile, Wang et al. reported that overexpression of miR-491 suppressed lung metastasis of OS cell in vitro and in vivo in addition it enhanced cisplatin (CDDP)-induced tumor growth inhibition and apoptosis [71]. Besides miRNAs, accumulated evidences indicated the significance of lncRNA in OS tumors. Gu et al. transfected lentivirus-mediated sh- LncRNA DICER1-AS1 or sh-control in MG-63 and U2OS cells, and injected them subcutaneously into the posterior flank of nude mice. They revealed that DICER1-AS1 knockdown inhibited tumor growth of osteosarcoma in vivo [72]. Chondrosarcoma (CS) has overall poor responses to conventional chemotherapy and radiotherapy due to resistant to radiation and chemotherapy [73]. Tang et al. reported miR-125 and miR-100 overexpression enhanced the sensitivity of CS to doxorubicin and cisplatin, respectively, which suggesting that ncRNA reduced antichemoresistance of CS cell [74]. Furthermore, cells transfected with pcDNABCAR4 or si-BCAR4 were injected into the nude mice, respectively. The result showed lncRNA BCAR4 knockdown decreased CS cell proliferation and migration in vivo in CS xenograft mouse [75]. Zhang et al. reported that miRNA let-7a mimic

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(simulate the miRNAs in vivo and enhance the function of endogenous miRNAs) could partly dampen let-7a-mediated CS suppression, and systemic delivery of synthetic let-7a to mice led to ESFT (Ewing sarcoma family tumor) inhibition in vivo [76, 77]. Multiple osteochondromatosis (MO) is an autosomal dominant disease [78– 80]. The molecular basis of MO genetic and clinical heterogeneity, including the causes underlying malignant transformation, is currently unknown. This leads to the lack of appropriate therapeutic strategy. Zuntini et al. investigated miRNA expression in osteochondroma and normal cartilage tissues to evaluate whether they could affect osteochondromas onset and/or clinical manifestations, such as has-miR-21, hsa-miR-140, hsa-miR-145, hsa-miR-214, hsa-miR-195, hsa-miR-199a, hsa-miR451, and hsa-miR-483. And results indicated that miRNAs differentially expressed in MO samples may hamper the molecular signaling responsible for normal differentiation of chondrocytes, contributing to pathogenesis and clinical outcome. Although further studies are needed to validate observations and to identify targets of miRNAs, this is the first study reporting on miRNA expression in growth plate and its comparison with pathological conditions [81]. In summary, ncRNAs play essential role in primary bone tumors. Therapeutic ncRNAs inhibit the proliferation and migration of tumor cells in vivo, and promote the apoptosis of tumor cells. At the same time, ncRNAs improve the sensitivity of cells to drugs as well. These ncRNAs provide potential possibility for primary bone tumors clinical treatment in the future.

8.4.2

The Therapy of ncRNA to Metastatic Bone Tumors

Bone metastases are responsible for the high morbidity in cancer patients with a strong clinical impact [82]. Metastases from carcinomas are the most common malignant tumors involving bone. Prostate, breast, and lung cancer are the common malignancies in adults, and are the most common tumors that metastasize to bone. Moreover, carcinoma of kidney, thyroid, and melanoma are other common tumors that metastasize to bone [83]. Bone is one of the preferred metastatic sites for lung cancer cells. So far, both accurate diagnosis and effective treatment of lung cancer bone metastases are difficult [84]. Gong et al. clarified that BC-5 miR-335 cells or control (SBC-5 Vector Ctrl) cells were injected intravenously into the tail vein of nude mice, and the results showed that miR-335 inhibited small cell lung cancer metastatic skeletal lesions [85]. Meanwhile, Liu et al. injected lentivirus vector carrying cells stably transfected with si-MALAT1 or the empty vector to mice. They found that knockdown lncRNA MALAT1 inhibited proliferation and induced apoptosis of tumor in vivo [86]. Brook et al. indicated miR-30 overexpression in estrogen receptor (ER)/progesterone receptor (PR) -negative breast cancer cells resulted in the reduction of bone metastasis burden in vivo and restored bone homeostasis [87]. MDA-MB-231 cells infected with lentiviruses carrying empty vector or miR-124 were injected through

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lateral tail vein of mice, and the results showed upregulation of miR-124 inhibited bone metastasis of breast cancer cells in vivo [88]. Unlike breast cancer, bone metastasis of prostate cancer (PCa) is mainly caused by osteogenic lesions [89]. More and more studies have proved miRNAs play the pivotal roles in bone prostate cancer bone [90, 91]. Tang et al. found a greatly suppressed incidence of PCa bone metastasis in vivo after agomir-133a-3p was injected into mice through tail vein [92]. Moreover, Huang et al. clarified that upregulated miR-141-3p dramatically reduced bone metastasis of PC-3 cells in vivo indicating that miR-141-3p mimics might represent a potential therapeutic avenue for the treatment of PCa bone metastasis [93]. It is reported that reintroduction of miR-148a, miR-34b, and miR-34c into cancer cells with epigenetic inactivation inhibited motility, tumor growth, and metastasis formation in xenograft models [94, 95]. Although there is still a long way to go before the use of ncRNA for bone tumor treatment clinically, researches on ncRNA help us to elucidate mechanisms of metastases bone tumors and facilitate the development of novel method to treat bone metastases of other tumors (Table 8.6). Although the treatment methods for various bone tumors are constantly updated, the overall survival rate of patients is still very low. At present, the roles of ncRNAs in bone tumors have been widely reported. The abnormal expression of ncRNA is closely related to the occurrence, development, and prognosis of bone tumors. In recent years, related researches have also increased. Although multiple experiments have shown that ncRNAs can inhibit the proliferation and metastasis of osteosarcoma in bone tumor treatment, the effective delivery of ncRNA and the screening of effective targets are still technical difficulties due to the harder bone tissue and the hydrophilic characteristics of ncRNA. Relevant teams are also actively researching these issues, and some novel delivery strategies have been developed, such as viral infections and nanoparticle delivery. Therefore, the research on ncRNA still has great research prospects and value for the treatment of bone tumors.

8.5

Conclusion

In the recent several decades, the roles and therapeutic effects of ncRNAs for bone diseases attracted more and more scientists’ attention. At the same time, bone disease research has become a very important matter in clinical medicine. However, the lack of clinical research is an important shortcoming of this research field, which resulted from the following reasons: (1) bone tissue has a lot of calcium salt deposition, leading to only few drugs can exert pharmacological actions, (2) the major concern for the application of ncRNA in bone disease treatment is the difficulty in delivering ncRNA into the bone tissue, which has a great impact on the efficacy of ncRNA, (3) expensive price of targeted delivery systems also limits the development of ncRNA treatment. Although several studies proved that ncRNA can attenuate bone disease symptoms remarkable in animal models via different deliver systems,

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Table 8.6 The therapeutics of ncRNA to bone tumor Drugs MiR-134

Formula Lentivirus

MiR-1433p MiR-449a and miR-424 SiLINC01116 MiR-20a

Lentivirus

Lentivirus

LINC00612

–

MiR-1545p mimics Circulating miR-25-3p

–

Lentivirus

–

Lentivirus

Administration Subcutaneously inject Subcutaneously inject Subcutaneously inject Inject into the proximal tibia Subcutaneously inject Subcutaneously inject Subcutaneously inject Subcutaneously inject

Period 3 weeks 4 weeks 13 days

6 weeks 30 days

3 weeks 3 weeks 10 weeks

MiRNA27a

The pCDH control vector

Subcutaneously inject

35 days

MiR-491

LOCK-iT pol II miR RNAi expression vector kit Lentivirus

Inject into the intramedullary cavity of the tibia

4 weeks

Subcutaneously inject Subcutaneously inject Subcutaneously inject Subcutaneously inject

30 days

MiR-363 MiRNA let-7a MiR-6245p MiR-4255p

– Lentivirus Lentivirus

5 weeks 28 days 6 weeks

MiR-2045p mimic

–

Subcutaneously inject

28 days

MiR-144

Lentivirus

30 days

MiR-15b mimics

Liposome

Intratumoral injection Tail vein injection

MiR-1908

Lentivirus

Subcutaneously inject

15 days

22 days

Efficacy Inhibited the growth of implanted OS tumors Inhibits tumor growth and metastasis in vivo Suppress osteosarcoma by targeting cyclin A2 expression Inhibited the tumor growth Reduces the proliferation of osteosarcoma cells in vitro and in vivo Promotes tumor growth of osteosarcoma Inhibited OS tumorigenesis in vivo Reflected tumor burden in both in vivo models and patients Inhibits cell invasion, migration, growth, and proliferation in osteosarcoma Inhibits osteosarcoma lung metastasis and chemoresistance

Induced the regression of osteosarcoma tumors. Inhibits osteosarcoma cell proliferation Promoted tumorigenesis and metastasis Suppressed OS cell proliferation, invasion, and migration Promotes apoptosis and inhibits migration of osteosarcoma Inhibited tumor growth and metastasis Modulates multidrug resistance in human osteosarcoma Has oncogenic, proliferation, migration, and invasion regulatory effects

Ref. [11] [96] [97]

[98] [68]

[99] [100] [101]

[102]

[71]

[103] [104] [105] [106]

[107]

[108] [109]

[110]

(continued)

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Table 8.6 (continued) Drugs MiR-8765p MiR-496

Formula Lentivirus –

–

Administration Subcutaneously inject Subcutaneously inject

6 weeks

MiR-210 mimics MiR-199a5p MiR-145

Lentivirus

MiR-23a

–

Subcutaneously inject

23 days

MiR-18a mimics

Lentivirus

Subcutaneously inject

5 weeks

MiR-5423p mimics

–

30 days

LncRNA NNT-AS1

Lentivirus

Injected into the tibial medullary cavity Subcutaneously inject

MiR-382

–

Subcutaneously inject

3 weeks

MiR-422a

–

6 weeks

MiR-203

–

Subcutaneously inject Subcutaneously inject

MiR-217

Lentivirus

MiR-124

–

LncRNA FOXD2AS1 LncRNA AFAP1AS1 MiR-143/ 145 mimics

Lentivirus

Inject into the tibial plateau

36 days

–

Subcutaneously inject

35 days

Lentivirus

Orthotopically inject

61 days

Lentivirus

Subcutaneously inject Subcutaneously inject Subcutaneously inject

Period 30 days

Subcutaneously inject Subcutaneously inject

30 days 30 days 42 days

5 weeks

4 weeks

3 weeks 24 days

Efficacy Inhibits cell proliferation, migration, and invasion Suppresses proliferation, invasion, migration, and in vivo tumorigenicity of human osteosarcoma cells Inhibits osteosarcoma growth Promotes tumor growth Inhibits tumor growth and metastasis in osteosarcoma Inhibition of miR-23a effectively reduced migration and invasion of osteosarcoma Inhibits cell growth and induces apoptosis in osteosarcoma Inhibits cell proliferation, migration, and invasion in osteosarcoma cells Suppresses cell proliferation and tumor growth in vitro and in vivo Inhibits tumor growth and enhance chemosensitivity in osteosarcoma Inhibits osteosarcoma proliferation Reduced cell growth in vitro and suppressed tumorigenicity in vivo Inhibit the proliferation, invasion, migration Suppresses growth and aggressiveness of osteosarcoma Inhibits cell proliferation, migration, and invasion in osteosarcoma Enhances cell proliferation and invasion in osteosarcoma Reduces the malignant properties of the chondrosarcoma

Ref. [111] [112]

[113] [114] [115]

[116]

[117]

[118]

[119]

[120]

[121] [122]

[123] [124]

[125]

[126]

[127]

(continued)

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Table 8.6 (continued) Drugs LncRNA BCAR4

Formula –

Administration Subcutaneously inject

Period 30 days

LncRNA HOTAIR and miR-4543p

–

Subcutaneously inject

30 days

MiR-29b3p mimics MiR-101 mimics

–

Intra-articular injection Tail vein injection

6 weeks

MiR-1295p mimics

MiR-181a mimics

Cotransfected with pCMV plasmids pcDNA3.1 vector

Lentivirus

MiR-199a mimics

6 weeks

Subcutaneously inject

24 days

Tail vein injection

6 weeks

Subcutaneously inject

7 days

MiR-206 mimics

Lentivirus

Subcutaneously inject

6 weeks s

MiR-381 mimics

–

Subcutaneously inject

6 weeks s

MiR-452 mimic

Lentivirus

Subcutaneously inject

4 weeks

MiR-494 mimics

–

Subcutaneously inject

30 days.

MiR-507 mimic MiR-519d mimic Anti-miR301a

–

Subcutaneously inject Intravenously inject Gluteal region injection

6 weeks s

Anti-miR20b

– PTEN expression vector TGFBR2 expression vector

Gluteal region injection

9 weeks 6 weeks

6 weeks

Efficacy Promoted chondrosarcoma cell proliferation and migration Mediated chondrosarcoma cell growth by negatively regulating miR-454-3p expression in chondrosarcoma Facilitates chondrocyte apoptosis and OA Inhibits chondrosarcoma metastasis in vitro and in vivo. Suppresses cell proliferation, migration and promotes apoptosis in chondrosarcomas Anti-angiogenic and antimetastatic effects of miR-181a inhibition Increased tumorassociated angiogenesis and tumor growth Enhances VEGF-A production in human chondrosarcoma cells and promotes angiogenesis Promotes VEGF-Cdependent lymphangiogenesis Promoted angiogenesis and tumor growth in human chondrosarcoma cells Inhibits migration and invasion of chondrosarcoma cells Inhibits tumor lymphangiogenesis Inhibits tumor metastasis Significantly suppressed Ewing’s sarcoma (ES) tumor growth in vivo Regulates cell cycle, apoptosis, and tumor proliferation

Ref. [75]

[128]

[129] [130]

[131]

[132]

[133]

[134]

[135]

[136]

[137]

[138] [139] [77]

[140]

(continued)

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Table 8.6 (continued) Drugs MiR-30a5p mimic MiR-124 mimic

Formula Lentivirus Lentivirus

Administration Subcutaneously inject Tail vein injection

Period 16 days 30 days

Anti-miR130b

–

Tail vein injection

6 weeks

MiR-138

–

Tail vein injection

6 weeks

MiR-1273p and miR-376a3p

–

Chorioallantoic membrane (CAM) of chicken eggs

7 days

LncRNA MALAT1

Lentivirus

Subcutaneously inject

18 days

LncRNA DANCR

Vector (pMSCV puro)

Injected subcutaneously or intravenously

4 weeks

MiR-30

Retroviral vector

Tail vein injection

28 days

MiR-466

Lentivirus

7 weeks

MiR-1273p

–

Tail vein injection Intratumoral injection

Agomir133a-3p MiR-1413p mimics

Retroviral vector Retroviral vector

Anti-miR210-3p

Retroviral vector

MiR-143 and -145

Luciferaselabeled vector PC-3

Tail vein injection Orthotopically implanted into the posterior prostatic lobe Inoculate into the left cardiac ventricle Tail vein injection

/

32 days 8 weeks

Efficacy Induces decreased cell proliferation and invasion Represses the mesenchymal features and suppresses metastasis in Ewing’s sarcoma Contributes to metastatic properties and phenotype seen in ES tumors. Inhibits metastatic potential in Ewing’s sarcoma cells The tumorigenicity of GCTSC is significantly reduced, resulting in lower tumor take rates and growth in vivo Significantly increased the migration, invasion, and tumorigenesis in vivo Working as a competitive endogenous RNA, promotes ROCK1-mediated proliferation and metastasis Inhibits breast cancer invasion, osteomimicry, and bone destruction Suppressed spontaneous metastasis to bone Suppresses PCa cell migration, invasion, and bone metastasis Represses the bone metastasis of PCa in vivo Mediates bone metastasis of prostate cancer via regulating the EMT

Ref. [141] [142]

[143]

[144]

[145]

[86]

[146]

[85]

[147] [70]

[87] [148]

10 weeks

Inhibits the bone metastasis of PCa in vivo

[149]

35 days

Mediates bone metastasis of prostate cancer via regulating the EMT

[92]

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clinical application is still a long way off. So, developing new deliver systems, which can apply in clinical, remains a challenging working. Acknowledgements This work was supported by the Natural Science Foundation of China (82072106, 32000924, and 81801871), the China Postdoctoral Science Foundation (2020 M683573, 2019 T120947, and 2017 M613210), the New Century Excellent Talents in University (NCET-12-0469), the Shenzhen Science and Technology Project (JCYJ20160229174320053), Shaanxi Provincial Key R&D Program (2018KWZ-10), Shaanxi Postdoctoral Science Foundation (2017BSHEDZZ13), Special Fund for Technological Innovation of Shaanxi Province (No. 2019QYPY-207), the Fundamental Research Funds for the Central Universities (3102018zy053).

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