Noncoding RNAs and Bone

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vi Preface

were considered to participate in a series of biological processes such as transcrip-

tion, 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 Tech-
nology 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, 2018SF-
263, 2018KA180038C180038, 2018JQ8032, 2018JQ3049, 2015JM3078, and
2015JQ3076), Shaanxi Provincial Key R&D Program (2018KWZ-10), Shaanxi
Preface vii

Postdoctoral Science Foundation (2017BSHEDZZ13), Special Fund for Technolog-

ical 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Xue Wang, Ruiyun Li, Xuechao Liang, Ye Tian, Airong Qian,
and Hui Li
2 MicroRNAs and the Diagnosis of Osteoporosis . . . . . . . . . . . . . . . . . 27
Ying Huai, Hui Li, Ye Tian, Airong Qian, and Zhihao Chen
3 MicroRNAs and Osteoarthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Shenxing Tan, Qian Huang, Xuechao Liang, Airong Qian, and Ye Tian
4 Mechanosensitive MicroRNAs and Bone Formation . . . . . . . . . . . . . 79
Zhihao Chen, Yan Zhang, Ying Huai, Fan Zhao, Lifang Hu, Chaofei
Yang, Ye Tian, and Airong Qian

Part II Long Noncoding RNAs and Bone

5 Roles and Mechanism of Long Noncoding RNAs in Bone Diseases . . 95
Dijie Li, Chaofei Yang, Ye Tian, Zhihao Chen, Airong Qian,
and Chong Yin
6 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

ix
x Contents

Part III RNA Synthesis Technology and RNA Therapy in Bone Diseases
7 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
8 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 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2 MiRNAs Involved in the Regulation of Osteoblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.3 MiRNAs Involved in the Regulation of Osteoclasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4 MiRNAs Involved in the Regulation of Bone-Related Signaling Pathways . . . . . . . . . . . . . . 11
1.4.1 MiRNAs Involved in the Regulation of Wnt Signaling Pathway . . . . . . . . . . . . . . . . . 11
1.4.2 MiRNAs Involved in the Regulation of BMP Signaling Pathway . . . . . . . . . . . . . . . . 16
1.4.3 MiRNAs Involved in the Regulation of TGF-β Signaling Pathway . . . . . . . . . . . . . . 17
1.4.4 MiRNAs Involved in the Regulation of RANK/RANKL/
OPG Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.4.5 MiRNAs Involved in the Regulation of M-CSF/c-FMS Signaling Pathway . . . . . 19
1.5 Conclusion and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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: wangxue1005@mail.nwpu.edu.cn; liruiyun@mail.nwpu.edu.cn;
lxc1204437320@mail.nwpu.edu.cn; tianye@nwpu.edu.cn; qianair@nwpu.edu.cn
H. Li (*)
Department of Adult Joint Reconstruction, Xi’an Hong-Hui Hospital, Xi’an, China

© Springer Nature Singapore Pte Ltd. 2021 3

A. Qian, Y. Tian (eds.), Noncoding RNAs and Bone,
https://doi.org/10.1007/978-981-16-2402-5_1
4 X. Wang et al.

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 30 untranslated region

ALK Activin receptor-like kinase
ALP Alkaline phosphatase
AP-1 Activator protein 1
APC Adenomatous polyposis coli
Bgn Biglycan
BMMs Bone marrow monocytes
BMPs Bone morphogenetic proteins
BMSC Bone mensencymal stem cell
CDC42 Cell division cycle 42
c-FMS Colony-stimulating factor-1 receptor
CTSK Cathepsin K
CXCL12 C-X-C motif chemokine ligand 12
DC-STAMP Dendritic cell-specific transmembrane protein
DKK1 Dickkopf-1
DKK2 Dickkopf-2
GSK3β Glycogen synthase kinase 3β
hADSCs Human adipose-derived mesenchymal stem cells
hBMSCs Human bone mesenchymal stem cells
HMGA2 High mobility group AT-Hook 2
HMOBs Human mandibular osteoblast-like cells
hMSCs Human mesenchymal stem cells
JNK c-Jun N-terminal kinase
LEF lymphoid enhancer-binding factor1
LRP Low density lipoprotein receptor-related protein
MAPK Mitogen-activated protein kinase
M-CSF Macrophage-colony-stimulating factor
MITF Microphthalmia-associated transcription factor
MMP Matrix metalloproteinase
MNGC Multinucleated giant cells
MSCs Mesenchymal stem cells
NFATC Nuclear factor of activated T cells C
1 MicroRNAs and Osteoporosis 5

NFATc1 Nuclear factor of activated T cells 1

OCN Osteocalcin
OPG Osteoprotegerin
OPN Osteopontin
OSX Osterix
PDCD4 Programmed cell death 4
PGC-1α Progastricsin 1α
PGE2 Prostaglandin E2
PMOP Postmenopausal osteoporosis
PPAR-gamma Peroxisome proliferator activated receptor gamma
PRKACB Protein kinase A catalytic subunit B
PTEN Phosphatase and tensin homolog
PVDF Poly (vinylidene-trifluoroethylene)/barium titanate
RANKL Receptor activator of nuclear factor Kappa-B ligand
rBMSCs Rat bone mesenchymal stem cells
RTK Receptor tyrosine kinase
Runx2 Runt-related transcription factor 2
SCD-1 Stearoyl-CoA desaturase 1
sFRP Secreted frizzled related protein
SIRT6 Sirtuin6
Smad SMA- and MAD-related protein
Sox6 SRY-box transcription factor 6
Spry1 Sprouty 1
SPRY2 Sprouty 2
TCF T cell-specific transcription factor
TGFBRI Transforming growth factor-β receptor I
TGF-β Transforming growth factor-beta
TNFSF13b Tumor necrosis factor superfamily member 13b
Tob1 Transducer of RbeB2 receptor tyrosine kinase 1
TRAF3 Tumor necrosis factor receptor associated factor-3
TRAF6 Tumor necrosis factor receptor associated factor-6
TRAP Tartrate-resistant acid phosphatase
Wnt Wingless-type MMTV integration site family members
XPO5 Exportin 5

1.1 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]. Emerg-
ing evidence suggests that it is a kind of multifactorial and complicated metabolic
6 X. Wang et al.

osteopathy, in which many kinds of cells in the bone microenvironment, especially

osteoblast and osteoclast, playing critical roles and coordinating the bone homeo-
stasis [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 environ-
mental 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 com-
plementary 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, differ-
entiation, 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.
1 MicroRNAs and Osteoporosis 7

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 signal-

ing 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 osteo-
porosis 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-
8 X. Wang et al.

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 homeo-
stasis and microstructure. Numerous studies have shown that dysfunction of osteo-
blasts 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 prolif-
eration, migration, and differentiation [11]. MiR-122 exerts inhibitory effect on both
proliferation and differentiation of osteoblasts in ovariectomized rats with osteopo-
rosis [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 differenti-
ation 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 mol-
ecules, 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 oste-
oblasts 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 tran-
scription 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 9

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 macrophage-
colony-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 glucocorticoid-
mediated 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 osteo-
clasts, as a miRNA that is significantly, differentially expressed throughout
osteoclastogenesis during miRNA profiling of monocyte-to-osteoclast differentia-
tion [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
10 X. Wang et al.

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 differen-
tiation 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 osteoclast-
specific 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-X-
C 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.
1 MicroRNAs and Osteoporosis 11

1.4 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 osteoclast-
related signaling pathways is increasingly evidenced. In this part, the roles of
miRNAs acting on the specific targets including vital signaling transduction mole-
cules, 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 embryogen-
esis 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 activa-
tion 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 intracellu-
lar signal transduction, interfering the ubiquitinoylation and proteasomal degrada-
tion of β-catenin result from the phosphorylation of axin/adenomatous polyposis coli
(APC)/glycogen synthase kinase 3β (GSK3β) complex and accumulating cytoplas-
mic 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
12 X. Wang et al.

Table 1.1 The roles of miRNAs involved in different bone-related signaling pathways
Effect on
bone
Signaling pathways miRNA Target Cell line/animal model formation
Wnt signaling pathway miR-376c Wnt-3 Calvarial osteoblast Inhibition
miR-22- Wnt-1 Clinical osteoporosis Inhibition
3p sample
miR-34a- Wnt-1 Clinical osteoporosis Inhibition
5p sample
miR-375- LRP5 Pre-osteoblasts Inhibition
3p
miR-545- LRP5 Pre-osteoblasts Inhibition
3p
miR-4739 LRP3 hBMSCs Inhibition
miR-27a APC Pre-osteoblasts Promotion
miR-26b GSK-3β rBMSCs Promotion
miR-139 β-Catenin hBMSCs Inhibition
miR-217 DKK1 MSCs Promotion
miR-483- DKK2 rBMSCs Promotion
3p
miR-128 DKK2 rBMSC Promotion
miR-96 SOST Ankylosing spondylitis Promotion
mice/ primary osteoblasts
Let-7c SCD-1 hADSCs Inhibition
BMP signaling miR-93 BMP2 Human femoral neck frac- Inhibition
pathway ture samples
miR-98 BMP2 hBMSCs Inhibition
miR-106a BMP2 MSCs Inhibition
miR-765 BMP6 hMSCs Inhibition
miR-450b BMP3 hADSCs Promotion
miR-494 BMPR2, Pre-myogenic C2C12 cells Inhibition
RUNX2
miR-1187 BMPR-II PMOP mice Inhibition
miR-203- SMAD1 rBMSCs Inhibition
3p
miR-135 SMAD5 Pre-osteoblasts Inhibition
miR-155 SMAD5 Pre-osteoblasts Inhibition
miR-185 BGN mMSCs/miR-185/ mice Inhibition
TGF-β signaling Let-7a-5p TGFBR1 mBMSC/PMOP mice Inhibition
pathway miR-10b SMAD2 hADSCs Promotion
miR-21 SMAD7 mMSCs Promotion
miR-130a SMURF2 BMSCs Promotion
miR-23a- PRDM16 Col1a1-miR23aC trans- Promotion
27a-24-2 genic mice
miR-140- TGFβ3 Pre-osteoblasts Promotion
3p
miR-34a RANKL Osteoclastss Promotion
(continued)
1 MicroRNAs and Osteoporosis 13

Table 1.1 (continued)

Effect on
bone
Signaling pathways miRNA Target Cell line/animal model formation
RANK/RANKL/OPG miR-212 RUNX2 hMSCs/PMOP mice Inhibition
signaling pathway miR-384 RUNX2 hMSCs/PMOP mice Inhibition
miR- PRKACB HMOBs Promotion
302a-3p
M-CSF/c-FMS signal- miR-214 PTEN BMMs Inhibition
ing pathway miR-142- PTEN BMMs Promotion
5p
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 postmen-
opausal 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 activa-
tion, 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 accumula-
tion and translocating into nucleus [63, 64]. MiR-139 negatively regulates osteo-
genesis 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 for-
mation 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.