LncRNAs as a new regulator of chronic musculoskeletal disorder

Abstract Objectives In recent years, long non‐coding RNAs (lncRNAs) have been found to play a role in the occurrence, progression and prognosis of chronic musculoskeletal disorders. Design and methods Literature exploring on PubMed was conducted using the combination of keywords 'LncRNA' and each of the following: 'osteoarthritis', 'rheumatoid arthritis', 'osteoporosis', 'osteogenesis', 'osteoclastogenesis', 'gout arthritis', 'Kashin‐Beck disease', 'ankylosing spondylitis', 'cervical spondylotic myelopathy', 'intervertebral disc degeneration', 'human muscle disease' and 'muscle hypertrophy and atrophy'. For each disorder, we focused on the publications in the last five years (5/1/2016‐2021/5/1, except for Kashin‐Beck disease). Finally, we excluded publications that had been reported in reviews of various musculoskeletal disorders during the last three years. Here, we summarized the progress of research on the role of lncRNA in multiple pathological processes during musculoskeletal disorders. Results LncRNAs play a crucial role in regulating downstream gene expression and maintaining function and homeostasis of cells, especially in chondrocytes, synovial cells, osteoblasts, osteoclasts and skeletal muscle cells. Conclusions Understanding the mechanisms of lncRNAs in musculoskeletal disorders may provide promising strategies for clinical practice.


| INTRODUC TI ON
Musculoskeletal disorders are a group of conditions that affect the motor system, including bones, muscles, tendons, ligaments and joints. 1 People with multiple disorders are particularly vulnerable, especially in the context of an ageing population. Musculoskeletal disorders include a variety of conditions such as osteoarthritis (OA), rheumatoid arthritis (RA), osteopenia, osteoporosis, fractures, sarcopenia, etc. 2 Non-protein-coding RNA makes up 98% of the whole human genome. 3,4 These functional RNAs can be divided into two groups according to the threshold of 200 nucleotides (NTS): small and long non-coding RNAs (lncRNAs). 5,6 LncRNAs regulate the activities of both nearby and distant genes by multiple mechanisms. It could act as a scaffold for transcription factors and other molecules involved in transcription initiation. 7 Moreover, it could serve as protein and microRNA decoys to interfere with cell division by regulating a series of key genes. 8 For those mainly located in the cytoplasm, it could directly target mRNA and induce translation. 9 Currently, an increased number of lncRNAs are found to be involved in the regulation of development and homeostasis of skeletal muscle system. 10,11 It is notable that lncRNAs take key roles in musculoskeletal disorders.
In this review, we summarized the functions and mechanisms of lncRNAs involved in the occurrence and progression of musculoskeletal disorders. Meanwhile, the potential of lncRNAs as promising targets for musculoskeletal disorders was also highlighted. An in-depth study of the pathological process, molecular regulatory mechanisms, cytokines and therapeutic targets of musculoskeletal disorders would greatly benefit patients before they progress to the end stage of the disease. We hope that this review will provide insight into the potential of lncRNAs as biomarkers and therapeutic targets for musculoskeletal disorders.

| Introduction of OA
OA, one of the most common musculoskeletal disorders, has been rising since the mid-20th century. It usually begins with agerelated degeneration of the articular cartilage surface, and its main pathological feature is cartilage destruction. At the joint level, pathogenic factors include joint injury, joint dislocation, abnormal joint loading and other factors. 12 It is well known that extracellular matrix (ECM) destruction, 13,14 inflammatory response and synovitis, 15,16 cell proliferation, 17 cell death (including apoptosis and autophagy) 18,19 and angiogenesis 20 are closely related to the pathological process of OA.
As early as in 2014, Xing et al reported the differentially expressed lncRNAs (73 up and 48 down) in OA cartilage compared with normal cartilage through microarray analysis. 21 Mounting studies have shifted from merely concentrating on the fate of articular cartilage to evaluating how the intra-articular microenvironment influences the occurrence and progression of OA. Detailed pathological process of OA is described in Figure 1. LncRNAs related to OA that have appeared in other literatures 22,23 will not be introduced in detail in this review. Together with the lncRNAs presented in this review, they are summarized in Table 1. This review mainly focuses on recent studies of lncRNAs.

| Role of lncRNAs in ECM degradation in OA
Articular cartilage is a type of connective tissue made up of chondrocytes. But, interestingly, chondrocytes make up only 1% of normal cartilage volume. The auto-synthetic ECM blocks the chondrocytes.
Nonetheless, they provide mechanical support for the cartilage and lubrication of the joint. It is also responsible for the composition and integrity of the matrix. 24 In OA chondrocytes, matrix metalloproteinases (MMPs) (including MMP-1 and MMP-13), metalloproteinase with a thrombospondin type 1 motif (ADAMTS) (including ADAMTS 1,4,5) and various types of disintegrin have been found to significantly increase the expression of matrix degrading proteins. 25 In addition, fibroblast-like synoviocytes (FLSs) has been reported to overexpress several enzymes (such as MMP-13) that degrade ECM. 26 Due to the unique composition of cartilage, ECM degradation is the most popular mechanism of OA associated with lncRNA. Recent studies have shown that lncRNA XIST (long non-coding RNA Xinactive-specific transcript) can be regarded as a star lncRNA. XIST was revealed to be upregulated in OA specimens and articular chondrocytes derived from OA tissue and IL-1β-treated articular chondrocytes (ACs). Downregulation of XIST suppresses the degradation of the ECM by binding a competing endogenous RNA (ceRNA) of miR-1277-5p. 27 LncRNA XIST is mainly localized in the nucleus and   (PGC-1β). 39 In OA, lncRNA IGHCγ1 was upregulated and was mainly Although chondrocytes proliferation is associated with natural regeneration, it may also lead to pathological processes. 46 Chondrocytes proliferate actively, causing some of them to grow and others to undergo hypertrophic changes to become hypertrophic chondrocytes. 47 On the molecular level, chondrocyte hypertrophy differentiation may also be characterized via high expression of collagen type X, MMP13 and Runt-associated transcription factor 2 (Runx2). Hyaline cartilage markers are decreased in the hypertrophic cells. It contains collagen type II, aggrecan and SOX9. 48 Some evidences indicate that OA-derived FLSs (OA-FLSs) play an important role in the proliferation, migration and apoptosis of chondrocytes. 49 Another report showed that overexpression of DNMT3A inhibited apoptosis and ECM degradation, but reduced miR-149-5p-induced cell viability promotion. This explains why XIST knockout can inhibit the development of OA through the miR-149-5p/DNMT3A axis. 57 The inhibition of SGTB expression by miR-142-5p could be relieved by lncRNA XIST. Therefore, the inhibition of miR-142-5p or the enhancement of SGTB can reverse the effects of XIST deletion on the growth and apoptosis of chondrocytes. 58 Exosomes are described as a kind of extracellular vesicles secreted by MSC in a resting state or under certain types of stress (such as hypoxia, radiation or oxidative damage), which can act as a messenger between MSC and differentiated cells, thereby inducing physiological changes. It has previously been reported that exosomes secreted from human MSCs promote cartilage regeneration.
Exosomes lncRNA KLF3-AS1 promote cartilage repair model of OA rats. 59 In another study by the same group, it is involved in apop- and OPN competed for the binding of miR-181c. Subsequently, the inhibitory effect of miR-181c on synovial cell proliferation and related factors inhibited by NEAT1 knockdown could be partially reversed. 56 In a novel study, researchers characterized the lncRNA expression profiles in human hyaline chondrocyte dedifferentiation, thereby identifying new potential mechanisms of chondrocyte dedifferentiation. It was found that AP001505.9 overexpression inhibited the dedifferentiation of chondrocytes. This discovery paves the way for further investigation into the mechanisms of dedifferentiation and OA treatment. 66

| Role of lncRNAs in angiogenesis in OA
The first step in ossification is vascular invasion, usually in nonvascular cartilage. The vascular system provides channels for different types of cells to participate in the recruitment of cartilage absorption and bone deposition. 67 VEGF is involved in vascular invasion of growth plate cartilage, hypertrophic cartilage remodelling and ossification of growth plate cartilage. 20 In the process of the development of OA, the level of MEG3 is negatively correlated with the level of VEGF, suggesting that MEG3 may regulate angiogenesis. 68 Currently, the importance of angiogenesis in the aetiology of OA has been demonstrated. It has been revealed that inhibition of angiogenesis may be a potential therapeutic target for OA by reducing OA-related pain and inflammation. 69,70

| LN CRNA S AND RHEUMATOID ARTHRITIS (R A )
RA is a chronic systemic autoimmune disease of unknown aetiology. In addition to the pro-proliferative and anti-apoptotic roles of exosomal NEAT1, the upregulation of NEAT1 promotes migration, invasion and inflammatory cytokine secretion in RA-FLSs. 81 Table 2, and the lncRNAs that appear in these reviews [87][88][89] are excluded, such as PISCAR, 90 LERFS 72 and NTT. 91

| LN CRNA S AND OS TEOP OROS IS
Osteoporosis results from the disruption of the balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Osteoporosis is a chronic systemic bone disorder characterized by loss of bone mass, microstructural destruction and increased fragility. One investigation found that more than one third of women over 50 years of age have osteoporosis, while only one fifth of men have osteoporosis, 92 indicating that women are at higher risk of osteoporosis than men. Before the age of 30, the process of bone loss begins. And it continues until death as a by-product of ageing.
Release of inflammatory factors such as TNF and IL-6 by senescence cells, as well as changes in the composition of bone marrow cells (osteoclast precursors, monocytes and granulocytosis), contributes to osteoporosis in the elderly. 93

| The role of LncRNAs in osteogenesis
Through competing endogenous RNA networks, we have identified functional lncRNAs in osteoblastic differentiation. 94 Long non-coding RNAs may serve as regulators of bone marrow stem cells (BMSCs) in osteoporosis. 95 The differentiation from MSCs to osteoblasts is a precise process regulated by multiple signalling pathways. [96][97][98] Many studies have shown that the expres- During this process, the expression of VEGF improves the coupling process of osteogenesis and angiogenesis. 106 Studies have revealed that MIR22HG expression is significantly reduced in BMSCs of osteoporotic mice and upregulated in hBMSCs during osteogenic differentiation. 107 In addition, a considerable quantity of literatures have described MSCs have the abilities not only in osteogenic differentiation, but also in adipogenic, myogenic and chondrogenic differentiation.

The basic pathogenesis of postmenopausal osteoporosis (PMOP)
is excessive bone resorption and insufficient bone formation due to oestrogen deficiency. 108

| The role of LncRNAs in osteoclastogenesis
Osteoclasts are multinucleated cells that originate from monocyte/ macrophage precursor cells and are responsible for bone resorption. 115 The regulatory roles of lncRNAs in osteoclasts have been less studied than those in osteoblasts. The first study that systemati- of bone marrow-derived macrophages (BMMs). Mice treated with BMMs plus EPC-derived exosomes showed increased neovascularization at the fracture site and enhanced fracture healing compared to mice treated with BMMs alone. 119 Similar to osteoblast, osteoclast activity was enhanced by BMSCS-derived exosome. MALAT1 from BMSCs-derived exosomes may be used as a miR-34c sponge to upregulate the expression of SATB2, contributing to the enhancement of osteogenic activity and the alleviation of osteoporosis symptoms in mouse models. 120 Studies in vivo and in vitro have shown that the expression of NEAT1 is closely related to the formation of osteoclasts. Mechanically, NEAT1 competitively binds to miR-7 and blocks its regulatory function of protein tyrosine kinase 2 (PTK2). Intergenic SNP rs12789028 acts as an allele-specific longrange enhancer of NEAT1 through chromatin interaction. 121 In order to prevent the degradation of its target gene Smurf2, lncRNA CCAT1 could competitively bind to miR-34a-5p. Inhibitory CCAT1 improved the pathological state of osteoporotic rats in vivo and restricted the osteocyte apoptosis of bone tissue in vivo. 122 Table 3 summarizes the lncRNAs introduced in this section.

| LN CRNA S AND G OUT ARTHRITIS (G A )
GA, the most common form of inflammatory arthritis, is caused by deposits of monosodium urate monohydrate (MSU) crystals in and around the joints. Elevated serum uric acid levels are considered to be an important risk factor for GA. 129 It is well established that MSU causes inflammation in the pathological process of gout, 130

| LN CRNA S AND K A S HIN -B ECK D IS E A S E (KBD)
KBD is an endemic, teratogenic osteochondropathy. Pathological features include degeneration and necrosis of articular cartilage and growth plates. [138][139][140] The aetiology of KBD is linked to environmental factors, 141,142 and hereditary factors are also thought to be involved. 143

| LncRNAs and Ankylosing spondylitis (AS)
AS is a systemic chronic disease with progressive development, characterized by chronic inflammatory responses in the sacroiliac joints and spine, and it belongs to RA. Several studies have shown that lncRNAs could be used as an independent diagnostic biomarker for AS, such as lncRNA AK001085, LINC00311, TUG1 and NKILA. [149][150][151][152] LncRNA MEG3 is a potential regulator in AS. It has anti-inflammatory effects, partly by targeting miR-146a. Overexpression of miR-146a reversed the inhibitory effect of abnormally expressed MEG3 on inflammatory factors. 153 Another study revealed that MEG3 promotes SOST expression to restrain the progression of AS by sponging let-7i. 154

| LncRNAs and Intervertebral disc degeneration (IDD)
Unlike articular cartilage, the intervertebral disc (IVD) is a wellwrapped and vascularless tissue that has three components: the nucleus pulposus (NP), annulus fibrosus (AF) and cartilaginous end plate (CEP). Nucleus pulposus is located in the centre of each disc and is highly hydrated and gelatinous, surrounded by the lateral annulus fibrosus. 159,160 IVD is the largest avascular structure in the body and the nerve endings only reach the inner ring. 161 Due to these structural characteristics, IVD is prone to degeneration. 162 At present, the aetiology of IDD is determined by genetic and environmental factors. Heredity is a major risk factor for IDD, as it is estimated that over 70% of cases are caused by genetics. 163,164 IDD is known to be driven by programmed cell death, 165  Here, we focus on the roles and functions of the newly discovered lncRNAs in IDD ( Figure 2). LncRNAs that appear in these published reviews 160,176 are not included in this section.

| LN CRNA S AND MUSCLE D IS E A S E S
Alterations in myogenesis and regeneration can lead to many muscle disorders (including muscle hypertrophy, muscular dystrophy and sarcopenia). Abnormal expression of lncRNAs is related to a variety of muscle diseases. Rescuing its normal expression in skeletal muscle can reduce the phenotype of the disease.

| LncRNAs in human muscle disease
Among all types of muscular dystrophy, one of the most common and severe disorders is Duchenne muscular dystrophy (DMD). DMD, Muscular atrophy is the most common muscle disorder in humans and is accompanied by myophagism and muscle weakness. 188 Li Lnc-ORA inhibited skeletal muscle myogenesis via regulating acting miR-532-3p/PTEN/PI3K/AKT axis. In addition, LNC-ORA interacted with IGF2BP2 (insulin-like growth factor 2 mRNA-binding protein 2) to influence the stability of myogenetic genes. 190 SMUL regulates myogenesis and muscle atrophy via TGFβ/Smad pathway.
The mechanism is SMUL's inhibition of Smurf2 production through NMD (nonsense-mediated mRNA decay). 191 Finally, miR22HG induces the maturation of miR-22-3p, which inhibits its target HDAC4 (histone deacetylase 4), thereby increasing downstream MEF2C (myocyte enhancing factor 2C), and ultimately promoting myoblast differentiation. 192 In conclusion, the current research focuses on muscle development after birth and growth, muscle hypertrophy and atrophy.
LncRNAs related to muscle hypertrophy and atrophy are summarized in Table 4. We excluded the lncRNAs that appeared in these published reviews. 11,193,194 In the near future, studies on muscle and lncRNAs will be oriented towards embryonic muscle generation and development, muscle fibre transformation, muscle function and movement, muscle ageing and metabolism, and muscle tumours.

| CON CLUS I ON S AND FUTURE PER S PEC TIVE S
In this review, we summarized the functions and regulatory mechanisms of lncRNAs involved in the occurrence and progression of musculoskeletal disorders. LncRNAs have been found to participate in the regulation of chronic musculoskeletal disorders under various pathological conditions. Current studies mainly point to the interaction axis between lncRNA and miRNA and downstream molecules.
More studies are urgently needed to investigate the underlying mechanism, such as the binding sites and ways of targeting downstream molecules, and whether there are multiple binding sites.
Furthermore, as described in this paper, some regenerative therapies involving stem cells are also associated with lncRNAs, such as mesenchymal stem cells (MSCs), which have been used in OA and IDD 195 therapy to assist tissue regeneration and exosome secretion.
This is also one of the research hotspots. And the role of lncRNAs in the regulation of intracellular or endochondral ossification and muscular dystrophy remains to be further studied. Finally, the interactions between circRNAs, lncRNAs, miRNAs and target genes also have considerable research potential.
Another area of active study is the post-transcriptional modifications of lncRNAs. Post-transcriptional modifications of RNA have been described in many sequencing-based transcriptome studies. Three major modifications include pseudouridine (Ψ), N6methyladenosine (m6A) and 5-methylcytosine (m5C). 196,197 Although the chemical modification of lncRNAs in other fields (eg, oncology) has suggested that its presence is important for the function of ln-cRNAs. But to date, no transcriptome changes have been reported to be associated with musculoskeletal diesease. Obviously, chemical modifications of RNAs are new areas of studying lncRNA functions.
Translational research on lncRNAs and musculoskeletal diseases will continue to flourish, in part due to our improving understanding of the functions of lncRNAs and the increasingly available practical methods to identify the functional domains of lncRNAs. Our understanding of the roles of lncRNAs in musculoskeletal disorders will lead to the development of new strategies to improve their clinical management.

ACK N OWLED G EM ENTS
This work was supported by grants from National Natural Science Foundation of China (81973606) and National Key Research and Development Program of China (2020YFC2004904).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.