Abbreviations ADSCs adipose-derived stem cells BMMSCs bone marrow mesenchymal stem cells BRWD1 bromodomain and WD repeat domain containing 1 CDH11 cadherin-11 ceRNA competing endogenous RNA circ-BRWD1 circRNA bromodomain and WD repeat domain containing 1 circRNAs circular RNAs ECM extracellular matrix ESCRT endosomal sorting complex required for transport EVs extracellular vesicles FLSs fibroblast-like synoviocytes GIT1 G-protein-coupled receptor kinase interacting protein-1 HDAC histone deacetylase IL interleukin IPFP infrapatellar fat pad lincRNA long intergenic noncoding RNA lncRNAs long non-coding RNAs MAPK6 mitogen-activated protein kinase 6 miRNAs microRNAs MMP13 matrix metalloproteinase 13 mRNAs messenger RNAs MSCs mesenchymal stem cells mTOR mammalian target of rapamycin MVBs multivesicular bodies ncRNAs non-coding RNAs NF-κB nuclear factor kappa-B NOX4 NADPH oxidase 4 OA osteoarthritis PCGEM1 prostate cancer gene expression marker 1 PTGS2 prostaglandin-endoperoxide synthase-2 RBP RNA-binding protein ROS reactive oxygen species RUNX2 runt-related transcription factor SFs synovial fibroblasts SMs synovial macrophages SOX9 sex-determining region Y-box 9 TGF transforming growth factor TLR toll-like receptor TNF tumour necrosis factor Tnfrsf21 tumour necrosis factor receptor superfamily member 21 TRAF6 TNF receptor-associated factor 6 UCMSCs umbilical cord mesenchymal stem cells UTR untranslated region 1 INTRODUCTION

Osteoarthritis (OA) is a prevalent degenerative disease involving the whole joint.1 It is characterized by morphological, biomechanical, biomolecular, and biochemical changes of cells and extracellular matrix (ECM) leading to progressive cartilage damage, synovial inflammation, subchondral bone sclerosis, and periarticular bone remodelling.2, 3 OA has an impact on the lives of 300 million people worldwide and carries a huge socioeconomic burden.4 At present, the treatment strategy for early and middle-stage OA is with or without pharmacological treatment to relieve joint pain.5 Surgical treatment is most widely used for late-stage OA patients with severe functional disability.6 Many repair methods, such as subchondral bone microfracture,7 subchondral bone drilling,8 allogeneic osteochondral transplantation,9 and autogenous osteochondral transplantation,10 have been proven to repair articular cartilage to varying degrees. However, there are few satisfactory strategies to improve cartilage homeostasis and delay OA progression since the specific pathogenesis of OA is still unclear. Understanding the underlying mechanisms of OA can facilitate the development of novel therapies for future clinical needs.

In recent years, extracellular vesicles (EVs) have emerged as a new method of cell-cell communication that participates in numerous physiological and pathological processes.11-14 EVs are membrane vesicles enclosed in a lipid bilayer and exist in almost all species.15, 16 EVs can be divided into three types, including exosomes, microvesicles and apoptotic bodies.17-19 As an important kind of EVs, exosomes have received the most attention over the past decade.20, 21 In 1981, Trams et al found that exfoliated membrane vesicles may serve a physiological function and suggested these vesicles as exosomes.22 In 1983, Harding et al found that membrane-bound vesicles could be released by multivesicular endosome exocytosis.23 In 1987, Johnstone et al observed that exosome release during reticulocyte maturation was related to plasma membrane activities.24 In 2006, Théry et al isolated exosomes from cell culture supernatants and found that the diameter of exosomes ranged from 30 to 150 nm.25 Usually, exosomes exhibit a cup-shaped or round morphology.26 In 2007, Valadi et al observed that exosomal messenger RNAs (mRNAs) and microRNAs (miRNAs) mediate intercellular communication, suggesting that exosomes play an important role as a cell–cell communication mediator.27 Exosomes are widely present in the human body fluids, including blood, urine, semen and other body fluids.11 They extend their life cycle by avoiding being swallowed by immune cells with their bilayer membranes and tiny volumes.20 On the outer membrane, exosomes contain phosphatidyl serine, such as biomarkers CD63, CD81, CD9, LAMP1 and TSG101.27 Exosomes rely on these membrane markers to pinpoint their destination via binding to specific receptors and then they release their cargoes in a targeted manner. Thus exosomes can be secreted by various cells and function as intercellular communication vehicles by delivering specific cargoes, such as protein,28 bioactive lipids,29 and nucleic acids,30 from the donor cells to the recipient cells through its paracrine activity.31

Non-coding RNAs (ncRNAs) are functional RNAs that can be transcribed but not translated.32-34 They are mainly comprised of miRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs).35, 36 Recent studies have revealed that ncRNAs modulate the expression of various target genes by regulation at post-transcriptional and post-translational levels, and are involved in multiple physiological and pathological processes, such as proliferation, apoptosis, differentiation, inflammation and homeostasis.37, 38 Recently, an increasing number of studies have shown that ncRNAs could be entrapped in and transferred between cartilage-related cells as exosomal cargoes.26, 39, 40 Meanwhile, the importance of the role of exosomal miRNAs, lncRNAs and circRNAs in OA progression has been gradually discovered. Exosomal ncRNAs (miRNAs, lncRNAs and circRNAs, etc.) are also expected to be an important way to explain the pathogenesis, diagnosis and cell-free treatment of OA.41 In this review, we summarize the biological functions of exosomal ncRNAs in OA and discuss the perspectives and challenges of exosomal ncRNAs application for OA patients in the future.

2 EXOSOMES BIOGENESIS

Exosomes biogenesis can be divided into different phases. First, the inward budding of the plasma membrane results in the formation of early endosomes. This process is also called endocytosis. Second, early endosomes mature into late exosomes by cargo selection. Third, several late endosomes accumulate to form multivesicular bodies (MVBs). Finally, the vesicular contents release by membrane fusion between MVBs and the plasma membrane and become exosomes (Figure 1).17, 20 Classical mechanisms for the formation and release of exosomes are driven by the endosomal sorting complex required for transport (ESCRT).42 ESCRT mainly consists of four complexes (ESCRT-0–III) with associated proteins as specific binding subunits.43 The ESCRT-III complex assembles on the endosome membrane and undergoes vesicle scission. Apoptosis-linked gene 2-interacting protein X, ATPase, and vacuolar protein sorting-associated protein regulate the ESCRT membrane-scission machinery.44 In addition, ESCRT-independent mechanisms have been proved in the absence of ESCRT. Lipids such assphingosine 1-phosphate and ceramidefunction in the ESCRT-independent pathway to regulate exosome release.45, 46 Most MVBs can be degraded by lysosomes.47 In some cases, the MVBs fuse with the cell membrane to release exosomes into the extracellular milieu through exocytosis.48 The target cells then can uptake exosomes by endocytosis, direct fusion or ligand–receptor binding.49, 50 These mechanisms may occur solely or in combination. In basic experiments, exosomes can be isolated and purified from cultured cell supernatants, serum or synovial fluid.51, 52 Exosomes can be injected intravenously or intraarticularly into study subjects for in vivo experiments,53 or added directly into the cell medium for uptake in vitro.54

Biogenesis and secretion of exosomes. Exosomes biogenesis can be divided into different phases. First, the inward budding of the plasma membrane results in the formation of early endosomes. This process is also called endocytosis. Second, early endosomes mature into late exosomes by cargo selection. Third, several late endosomes accumulate to form multivesicular bodies (MVBs). Finally, the vesicular contents release by membrane fusion between MVBs and the plasma membrane and become exosomes. The recipient cells then can uptake exosomes by endocytosis, direct cell membrane fusion or ligand-receptor binding

3 EXOSOMES IN OA

Studies on the roles of exosomes in OA have gained increasing attention. To date, there are two main research directions in this field. Some researchers focused on the diagnostic value and biological functions of exosomes in OA, and others focused on the therapeutic effects of exosomes on OA. The exosomes can be detected in the articular cavity during OA progression. In addition, the exosomes are derived from various joint cells including chondrocytes,55 osteoblasts,56 synovial mesenchymal stem cells (MSCs),53 fibroblasts,57 infrapatellar fat pad MSCs,58 tenocytes,59 and tendon stem cells.60 The exosomes could mediate cell-cell communications and regulate diverse cell phenotypes including cell proliferation, migration, differentiation, autophagy, matrix synthesis, inflammatory response, bone remodelling and cartilage degeneration during OA progression.5 The exosomal contents in OA are the basis of the physiological function of n exosomes in intercellular communication and signal transduction.61 The contents, including miRNAs, lncRNAs, DNA, lipid and protein, vary with disease progression and contribute to the pathological changes in disease.33, 34 To date, an increasing number of studies have demonstrated the significant role and underlying mechanism of exosomal contents in the OA process, including miRNAs, lncRNAs, circRNAs and proteins. In addition, Davies et al found that exosome profile and protein content from OA osteoblasts were temporally regulated during osteogenesis in bone remodelling.56 However, DNA, other types of RNA and the lipid content of exosomes in OA pathology have not been reported and require further investigation.

4 ncRNAs AND EXOSOMAL ncRNAs

Though different in location, structure, length and function, ncRNAs consist of approximately 98% of all transcriptional outputs.62 These ncRNAs exert an effect on normal gene expression and disease progression, making them new targets for mechanism evaluation and therapeutic potential.63

MiRNAs are multifunctional ncRNAs with 22-25 bases encoded by endogenous genes. MiRNAs regulate the stability and translation of mRNAs. They can bind to the 3′-untranslated region (UTR) of target mRNAs to regulate downstream pathways.64

LncRNAs are ncRNAs with a length >200 nt and participate in multiple cellular processes through interacting with miRNAs.65 According to their positions relative to the protein-coding genes, lncRNAs can be divided into five categories: (a) sense lncRNA, (b) antisense lncRNA, (c) bidirectional lncRNA, (d) intronic lncRNA, or (e) intergenic lncRNA.65, 66 Due to the flexibility of RNA, lncRNAs can fold into a unique secondary conformation, including DNA binding domains, RNA binding domains and protein binding domains, which enables them to form a broad regulatory network with DNA, RNA and protein complexes, and participate in the regulation of multiple gene expression. Salmena et al first proposed the concept of competing for endogenous RNA (ceRNA).67 The ceRNA, which is also called ‘miRNA sponge’, is a new mechanism of lncRNA. LncRNA can bind and isolate miRNA as ceRNA away from the sites that act on mRNA, thereby reducing the effect of miRNA on mRNA expression.68 Pearson et al explored for the first time the changes in lncRNA levels associated with the inflammatory response in human chondrocytes. They indicated that the inflammatory response in human OA chondrocytes was associated with widespread changes in the profile of lncRNAs, including the long intergenic noncoding RNA (lincRNA) p50-associated cyclooxygenase 2-extragenic RNA and two novel chondrocyte inflammation-associated lincRNAs (CILinc01 and CILinc02).69

CircRNA is a type of ncRNA with a closed circular structure. The biogenesis of circRNA follows the back splicing mechanism. The formation of a circRNA often requires the donor splice site to be conjugated with an upstream acceptor site.70 Three mechanisms have been proved as the biogenesis of circRNAs: intron pairing-driven circularization, RNA-binding protein (RBP)-driven circularization, and lariat-driven circularization.71 The biological functions of circRNA include: (a) acting as miRNA sponges; (b) acting as RBP-related protein sponges or decoys; and (c) translating circRNA peptides under certain circumstance.72

The miRNAs and other ncRNAs have been proposed as the most related cargo in the exosomes as a small number of molecules are able to affect diverse proteins or enzymes in cellular pathways in the targeted cells.73 Unlike intracellular RNAs, exosomal ncRNAs are more stable because of their bilayer membrane structure and ability to bind to RBP. Exosomes prevent ncRNAs as cargoes from degradation by ribonucleases extracellularly and therefore enable the clinical application of exosomal ncRNA.41, 74

Exosomal ncRNAs are transferred to recipient cells and exert roles in terms of chondrogenesis, ECM formation, apoptosis, proliferation, migration and inflammation (Figure 2).

The ncRNAs regulate the pathological process of osteoarthritis (OA). Recipient cells uptake exosomes carrying ncRNAs. Following released as cargoes, ncRNAs regulate the pathological process of OA, through proliferation, migration, chondrogenesis, extracellular matrix (ECM) formation, chondrocyte differentiation, apoptosis, and inflammation. miRNA can bind mRNA to modulate its expression. However, lncRNA and circRNA can act as the sponge for miRNA to alleviate the regulation of miRNA on the expression of downstream genes

5 EXOSOMAL miRNAs IN OA

In 2007, Valadi et al found that miRNAs in exosomes could be delivered into recipient cells, and function in the cells. They proposed that this RNA should be called ‘exosomal shuttle RNA’.27 In past years, the exosomal shuttle miRNAs were found to play important roles in cartilage homeostasis and OA progression via multiple mechanisms. The biological functions of exosomal miRNAs reported in previous studies are shown in Table 1.

TABLE 1. Exosomal miRNAs and their biofunction in osteoarthritis Exosome source Model Cargo Target cells Target genes Signalling pathway Function First author and year Synovial fluid Human miR-185-5p Chondrocytes Estrogen TLR signalling pathway Suppress chondrocyte/chondrogenesis Kolhe 201751 miR-7107-5p Signalling genes Promote inflammation Synovial MSCs Rat miR-140-5p Chondrocytes RalA No referred Promote ECM secretion, proliferation and migration Tao 201753 Human MSCs Mice miR-92a-3p Chondrocytes WNT5A Wnt signalling pathway Promote proliferation and ECM formation Mao 201875 Suppress cartilage degradation Primary chondrocyte Human miR-95-5p MSCs HDAC2/8 No referred Promote chondrogenesis Mao 201876 Human MSCs In vitro miR-193b-3p Chondrocytes HDAC3 No referred Promote chondrogenesis and cartilage formation Meng 201877 Human BMMSCs Human miR-320c Human MSCs/chondrocytes SOX9, MMP13 No referred Promote chondrogenesis Sun 201926 IPFP MSCs Mice miR-100-5p Chondrocyte mTOR mTOR-autophagy signalling pathway Maintain cartilage homeostasis Wu 201958 Suppress apoptosis Human chondrocyte In vitro miR-8485 Human BMMSCs GSK3B, DACT1 Wnt/β-catenin signalling pathway Promote chondrogenesis Li 202078 Human UCMSCs In vitro miR-381-3p Human UCMSCs TAOK1 Hippo signalling pathway Promote chondrogenesis Jing 202079 FW method synthesis Rabbit miR-140 Rabbit BMMSCs SOX9 No referred Promote chondrogenesis Won Lee 202080 Human BMMSCs Rat miR-26a-5p SFs PTGS2 No referred Promote apoptosis Jin 202081 Suppress proliferation, migration and inflammation Human BMMSCs Mice miR-136-5p chondrocyte ELF3 No referred Promote migration and ECM formation Chen 202082 Suppress ECM degeneration Mice BMMSCs Mice miR-210 Chondrocyte Tnfrsf21 NF-κB signalling pathway Promote proliferation He 202083 Suppress apoptosis Osteoclast Mice let-7a-5p chondrocyte Smad2 No referred Promote hypertrophic differentiation Dai 202084 BMMSCs Rat miR-127-3p chondrocyte CDH11 Wnt/β-catenin signalling pathway Suppress chondrocyte damage Dong 202185 BMMSCs Rat miR-135b SMs MAPK6 No referred Promote M2 polarization of SMs and cartilage repair Wang 202186 Human UCMSCs In vitro miR-100-5p Chondrocytes NOX4 No referred Suppress apoptosis Li 202187 Human BMMSCs Rat miR-361-5p Chondrocytes DDX20 NF-κB signalling pathway Suppress OA damage Tao 202188 SFs Rat miR-126-3p Chondrocytes No referred Multiple signalling pathways Promote proliferation and migration Zhou 202152 Suppress inflammation, apoptosis and cartilage degeneration Abbreviations: ADSCs, adipose-derived stem cells; BMMSCs, bone marrow mesenchymal stem cells; CDH11, Cadherin-11; DDX20, Asp-Glu-Ala-Asp-box polypeptide 20; ECM, extracellular matrix; FW, freeze and thaw; HDAC, histone deacetylase; IPFP, infrapatellar fat pad; MAPK6, mitogen-activated protein kinase 6; MMP13, matrix metalloproteinase 13; MSC, mesenchymal stem cells; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor kappa-B; NOX4, NADPH oxidase 4; PTGS2, prostaglandin-endoperoxide synthase-2; SFs, synovial fibroblasts; SMs, synovial macrophages; SOX9, sex-determining region Y-box 9; TLR, toll-like receptor; Tnfrsf21, tumour necrosis factor receptor superfamily member 21; UCMSCs, umbilical cord mesenchymal stem cells.

Cell loss is one of the main pathogenic factors of OA. Therefore, cell proliferation and migration can improve the OA process and cartilage repair.89 Associated genes typically participate in cell proliferation and migration to regulate cell growth and motility. Exosomal miRNAs are involved in this process. There have been a few studies on the role of human bone marrow MSCs-derived exosomes overexpressing miRNAs in OA. For instance, the exosomal miR-92a-3p derived from human bone marrow MSCs has a more potent effect on chondrocyte proliferation and motility enhancement than the MSCs exosomes. Luciferase reporter assay has demonstrated that exosomal miR-92a-3p inhibits WNT5A expression in both MSCs and primary human chondrocytes.75 Similarly, Sun et al found that exosomes derived from human bone marrow MSCs overexpressing miR-320c were more effective than exosomes derived from human bone marrow MSCs at increasing cell motility.26 Chen et al have suggested that migration of chondrocytes is promoted by exosomal miR-136-5p from bone marrow MSCs.82 Likewise, exosomes derived from miRNA-210-overexpressing bone marrow MSCs improve chondrocyte proliferation.83 In addition, a recent study conducted by Jin et al showed that human bone marrow MSC-derived exosomes could transfer miR-26a-5p into synovial fibroblasts. Human bone marrow MSC-derived exosomes overexpressing miR-26a-5p weaken synovial fibroblasts proliferation.81

In addition to bone marrow MSCs-derived exosomes, some studies have focused on exosomes from different kinds of cells. Exosomes from miR-140-5p-overexpressing synovial MSCs can enhance the proliferation and migration of chondrocytes without decreasing ECM secretion. The exosomal miRNAs are transferred into receptor cells and activate Yes-associated protein via the Wnt signalling pathway.53 In addition, exosomes derived from miR-126-3p overexpressing synovial fibroblasts can also enhance chondrocyte migration and proliferation.52

OA is characterized by chronic cartilage degeneration. Thus, the studies on exosomal miRNAs in OA have focused on the roles of miRNAs in chondrogenesis, chondrocyte differentiation induction and ECM formation or degradation. In the study by Mao et al exosomal miR-92a-3p was upregulated 7.89-fold in exosomes after chondrogenic differentiation of MSCs. Further investigation revealed that exosomal miR-92a-3p may target WNT5A to promote sex-determining region Y-box 9 (SOX9) and aggrecan expression and enhance cartilage development.75 Similarly, the same authors in their another study treated chondrocytes with 50 μg/mL exosomes derived from miR-95-5p-overexpressing primary chondrocytes. They found that exosomal miR-95-5p could mediate cartilage-specific gene expression in chondrocytes. Exosomes derived from miR-95-5p-overexpressing primary chondrocytes could enhance chondrogenesis and maintain cartilage homoeostasis by targeting histone deacetylase (HDAC) 2/8.76 Furthermore, exosomes derived from human bone marrow MSCs overexpressing miR-320c have been found to downregulate matrix metalloproteinase 13 (MMP13) expression and upregulate of SOX9 and CoL2A1 expression during human bone marrow MSC chondrogenic differentiation.26 In addition, Jing et al suggested that kartogenin-preconditioned small EVs induced human umbilical cord MSCs to differentiate into chondrocytes in rabbits with full-thickness cartilage defects. This induction was attributed to the exosomal miR-381-3p through direct suppression of TAOK1 via the Hippo signalling pathway.79 In addition to MSC-derived exosomal miRNAs, Li et al found that chondrocyte-derived exosomal miR-8485 regulated the Wnt/β-catenin pathways to promote chondrogenic differentiation of bone marrow MSCs. Mechanistically, exosomal miR-8485 represses GSK-3b expression by targeting GSK3B, and induces p-GSK-3b (Ser9) by targeting DACT1 via activating the Wnt/β-catenin pathways.