Promising diagnostic and therapeutic circRNAs for skeletal and chondral disorders

Circular RNAs (circRNAs) belong to a highly conserved subtype of non-coding RNAs, produced by the back-splicing of specific regions of pre-mRNA. CircRNAs have wide-ranging effects on eukaryotic physiology and pathology by acting as transcription regulators, miRNA sponges, protein sponges, and templates for translation. Skeletal and chondral disorders are the leading causes of pain and disability, especially for elders, affecting hundreds of millions of people worldwide. Plenty of evidence have shown that circRNAs are dysregulated and play vital roles in the occurrence and progression of skeletal and chondral disorders. Herein, we systematically summarize the emerging roles and underlying molecular mechanisms of hub circRNAs in the pathogenesis of several representative skeletal and chondral disorders. Our findings may provide further insight into the mechanistic details of the role of circRNA in bone or cartilage metabolism, and highlight the promising application of circRNAs in serving as potential diagnostic or therapeutic targets for the prevention and treatment of skeletal and chondral disorders.


Introduction
Circular RNAs (circRNAs) are recently discovered non-coding RNAs (ncRNAs) that consist of a specially covalently closed ring structure and can be stably expressed in multiple cell lines [1,2]. CircRNAs derive from canonical splice sites, upon suppression or slowing down of the splicing of pre-mRNA [3,4]. CircRNAs are classified based on their sequence. There three types of circRNAs are exonic circRNAs (EcRNAs), intronic circRNAs (ciRNAs) and exon-intron circRNAs (EIcRNAs) ( Figure 1A) [5,6]. EcRNAs are mainly found in the cytoplasm, while ciRNAs and EIcRNAs are predominantly found in the nucleus. EcRNAs are the most abundant circRNAs and account for over 80% of the known circRNAs [1,7,8].
CircRNAs were first discovered in pathogens in the 1970s [9] and were initially thought to be transcriptional splicing intermediates or byproducts of pre-mRNA splicing errors [10,11]. Since then, due to the advancements in RNA sequencing technologies and bioinformatic analyses, thousands of circRNAs have been identified [12]. Multiple lines of evidence have highlighted the significant impact of circRNAs on various eukaryotic physiological and pathological processes [13]. CircRNAs in the nucleus (ciRNAs and EIcRNAs) play roles in host gene transcription regulation ( Figure 1B) [5,14,15], while cytoplasmic circRNAs have been shown to function as miRNA sponges, thereby promoting the expression of downstream mRNAs ( Figure 1C) [7,16,17]. Recently, several circRNAs have been implicated in the function and localization of proteins by serving as a decoy, and facilitating protein folding and recruitment ( Figure  1D) [7,[16][17][18][19][20]. Some studies have also shown that Ivyspring International Publisher coding circRNAs can play critical roles in human diseases ( Figure 1E) [21][22][23][24].
CircRNAs function as pervasive regulators of cellular and physiological processes and have been proven to be vital in many diseases, such as skeletal and chondral disorders [25][26][27]. Skeletal and chondral disorders are among the leading debilitating factors and represent an increasing societal and economic burden in the context of aging population and increasing life expectancy. The most common of these diseases are osteoarthritis, osteonecrosis of the femoral head (ONFH), osteoporosis, and rheumatoid arthritis (RA), affecting hundreds of millions of people worldwide [28][29][30][31]. Several factors have been associated with the occurrence and progression of skeletal and chondral disorders, including inflammation, apoptosis, degradation of extracellular matrix (ECM), and an imbalance between osteogenesis and adipogenesis [30,[32][33][34][35][36]. However, the underlying molecular mechanisms of the pathology of these diseases still remains elusive, and an understanding of these mechanisms is essential for both the prevention and treatment of bone and cartilage disorders. Studies have begun to highlight the differential expression of circRNAs in bone and cartilage diseases as well as their regulatory roles [37]. Further characterizing the roles of circRNAs in the pathological processes of skeletal and chondral disorders could provide new avenues for both diagnosis and treatment.
In this review, we summarize recent studies of circRNAs involved in common skeletal and chondral disorders, such as osteoarthritis, ONFH, osteoporosis, and RA, and highlight the potential applications of these circRNAs in the prevention and treatment of these disorders.

CircRNA and osteoarthritis
Osteoarthritis is recognized as the most common disease of the musculoskeletal system, with up to 250 million cases globally [38]. Cartilage damage associated with osteoarthritis can be caused by mechanical, inflammatory, or metabolic stresses that alter cartilage properties [39][40][41]. As a result, hypertrophic chondrocytes attempt to repair eroded cartilage, but in turn release more matrix degradation products and proinflammatory factors, which then leads to structural alterations in articular cartilage, subchondral bone, synovium, capsule, ligaments, and periarticular muscles [42][43][44]. Here, we discuss osteoarthritis-related circRNAs and their regulatory effects on inflammatory and matrix metabolic factors in osteoarthritis-related cells. Understanding the role of these circRNAs can reveal new strategies for intervening the progression of osteoarthritis.

Diagnostic circRNAs of osteoarthritis
CircRNAs have been widely recognized as stable biomarkers due to their resistance from degradation by RNases [45]. The use of diagnostic circRNAs in peripheral blood and synovial fluid could have a significant impact on osteoarthritis diagnosis. Studies have indicated that hsa_circ_0104873, hsa_circ_ 0104595, and hsa_circ_0101251 are steadily expressed in synovial fluid, while hsa_circ_0032131_CBC1 is significantly over-expressed in the blood samples of osteoarthritis patients (Table 1) [46,47]. Further investigation of these circRNAs could set the groundwork for the advancement of minimally invasive, highly specific, easily accessible, and rapid diagnostic biomarkers.
Additionally, synovitis is the main cause of joint pain in osteoarthritis [70]. CircGCN1L1 was found upregulated in the synovium and play its roles by promoting synoviocyte proliferation and chondrocyte apoptosis in osteoarthritis. Silencing of circGCN1L1 attenuates the loss of condylar cartilage and subchondral bone via the circGCN1L1-miR-330-3p-TNF axis ( Figure 2B) [71].
These osteoarthritis-related circRNAs may function as novel therapeutic targets for the treatment and prevention of osteoarthritis. Future work exploring the upstream regulation of aberrantly expressed circRNAs in osteoarthritis, as well as the molecular details of circRNA-protein interactions in osteoarthritis, will be required to further understand the therapeutic potential of osteoarthritis -related circRNAs.

CircRNAs and ONFH
Osteonecrosis is a class of orthopedic diseases that is caused by the interruption of blood flow, affecting over 20 million people worldwide [72][73][74]. Due to its anatomical structure, the femoral head is particularly likely to undergo osteonecrosis [75]. Common risk factors of ONFH include excessive use of steroids or alcohol, trauma, or sickle cell anemia [76], all of which reduce blood supply to the femoral head, thus resulting in bone necrosis and alteration [77]. Important to the prevention of bone necrosis is bone regeneration via the differentiation and proliferation of bone marrow stromal cells (BMSCs) [78]. However, an imbalance of osteogenesis and adipogenesis of BMSCs has been observed to disrupt bone remodeling. Studies have revealed that circRNAs are dysregulated during osteogenesis. Here, we review circRNAs that have been shown to play critical roles in regulating osteogenesis and adipogenesis, to shed light on a regulatory mechanism for steroid-induced osteonecrosis of the femoral head (SONFH).
The correlation between circRNAs and ONFH is still in its infancy. To date, studies of the ONFH-related circRNAs have mainly highlighted their roles in osteogenesis or adipogenesis of BMSCs, the imbalance of which has a critical role in the progression of ONFH (Table 2) [36,79,80]. By screening the circRNA expression profiles in BMSCs from patients with SONFH, and using bioinformatics and functional characterization assays, CDR1as was found up-regulated in SONFH-BMSCs, and thus could play a critical role in osteogenic/adipogenic differentiation disorders via the miR-7-5p/WNT5B axis of regulation (Figure 3) [36]. Kuang et al. identified circUSP45 as an upregulated circRNA in BMSCs isolated from SONFH patients [80]. RNA pull-down and dual luciferase reporter assays were performed to confirm that circUSP45 mainly localizes in the cytoplasm and directly interacts with miR-127-5p. Further experiments verified that circUSP45 upregulates the expression of PTEN and inhibits AKT pathway by sponging miR-127-5p, thereby suppressing the expression of osteogenic genes, such as bone morphogenetic protein-2 (BMP2) and runt-related transcription factor 2 (RUNX2). Additionally, the anti-bone metabolism function of circUSP45 was verified in vivo by a SONFH rat model.   Therefore, silencing CDR1as and circUSP45 may promote bone metabolism and improve bone mass in SONFH progression. However, future work is required to understand more mechanistic details of regulatory circRNAs and their roles in the pathogenesis of ONFH (Figure 3).

CircRNAs and osteoporosis
Osteoporosis is a systemic and metabolic skeletal disorder affecting with more than 200 million individuals affected worldwide [81,82]. Osteoblastic bone formation and osteoclastic bone resorption dynamically maintain bone homeostasis [83]. Osteoporosis is typically characterized by decreased osteoblastic activity and increased osteoclastic activity [84]. During osteoporosis, BMSCs, which are precursors to osteoblasts, have been shown to have a lower osteogenic differentiation potential [85]. Regulatory mechanisms underlying osteoporosis are complex and involve a number of pathways, including circRNA mediation pathway.

Diagnostic circRNAs of osteoporosis
Here, we summarized circRNAs that are abnormally expressed in human bodily fluids from osteoporosis patients. These circRNAs may be promising candidates for clinical diagnosis of osteoporosis. In particular, circ_0002060 was shown by Huang et al. via microarray and bioinformatic analyses, to be upregulated in clinical samples, suggesting its use as a potential biomarker (Table 1) [86,87].
Some studies have also shown the regulatory mechanism of circRNAs in other types of stem cells. The effect of circ_0026827 on human dental pulp stem cells (DPSCs) during osteogenesis has been examined by Ji et al. [102] to seek novel therapeutics for osteoporosis. These studies showed that circ_0026827 function as a sponge of miR-188-3p to promote osteoblastic differentiation of DPSCs via the upregulation of Beclin-1-mediated autophagy and RUNX1 signaling pathways. CircFOXP1 were found to play critical roles in promoting osteogenic differentiation of adipose-derived mesenchymal stem cells (ADMSCs) in osteoporosis by targeting miR-33a-5p. In vivo and in vitro evidence indicates circFOXP1 could enhance the expression of FOXP1, thereby promote the osteogenic differentiation of ADMSCs and bone formation in osteoporosis ( Figure  4A) [103].
To date, circRNA_28313 is the only circRNA that has been shown to be a regulator of osteoclast resorption. Recent studies by Chen et al. [104] have examined mechanisms, involving circRNA regulation, of excessive bone resorption caused by osteoclasts during their differentiation. The authors used RANKL+CSF1-treated bone marrow monocyte/ macrophage (BMM) cells to mimic osteoclasts during the progression of osteoporosis. Their results showed that circRNA_28313 is dramatically overexpressed upon RANKL+CSF1 stimulation, while miR-195a is down-expressed.
Further bioinformatics and experimental validation both in vitro and in vivo showed that circRNA_28313 interacts with miR-195a and consequently regulates the colony-stimulating factor (CSF1) gene. High expression level of CSF1 in turn, promotes the expression of downstream proteins such as PU.1 (a factor related to osteoclast differentiation), nuclear factor of activated T cells c1 (NF-ATc1), tartrate-resistant acid phosphatase (TRAP), and Cathepsin K (CTSK), thereby enhancing osteoclast-induced bone absorption ( Figure 4B).
Thus, circRNAs can serve as powerful therapeutic candidates to promote bone regeneration and thus reverse the progression of osteoporosis. Moreover, a deeper understanding of these circRNAs can shed light on the mechanisms underlying regulation of circRNAs in bone metabolism.

CircRNAs and rheumatoid arthritis
Rheumatoid arthritis (RA) is a common chronic autoimmune disease characterized by inflammatory destruction that can cause serious cartilage and bone damage, affecting approximately 1% of the population worldwide [30]. Several non-coding RNAs have been identified as regulators of RA via different pathways, but the exact mechanisms underlying the role of circRNAs in RA remain to be understood.

Diagnostic circRNAs of rheumatoid arthritis
Early diagnosis is critical to optimal therapeutic success for rheumatoid arthritis (RA). Existing studies have identified a several circRNAs that could serve as diagnostic biomarkers of RA, partially due to their increased stability in plasma, serum, or other biofluids [105]. Here, we summarizes potential biomarkers identified by various studies (Table 1) [106][107][108][109][110][111].

Regulatory circRNAs of rheumatoid arthritis
The main pathological features of RA are autoimmune response and inflammation [112].
Recently, a number of circRNAs have been implicated in RA pathogenesis (Table 2), but their function and hidden molecular mechanism in immune and inflammation regulation still remains little known [30].
Given that the phosphatidylinositol-3-kinase/ AKT/mTOR (PI3K/AKT/mTOR) signaling pathway plays a crucial role in cellular proliferation and inflammatory responses [113,114], circFADS2 and ciRS-7 have been implicated in the initiation and progression of RA by regulating these pathways. CiRS-7 promotes the inflammation of PBMCs by sponging miR-7 to upregulate mTOR while circFADS2 protects LPS-treated chondrocytes (RA model cells) from apoptosis by mediating mTOR expression via sponging miR-498 ( Figure 5A and B) [115][116][117]. CircRNA_09505 is an up-regulated circRNA which can promote AKT1 expression via miR-6089/IκBα/NFκB signaling pathway in macrophages, thereby aggravating inflammation and joint damage in RA ( Figure 5B) [118].  Joint swelling and pain reflect synovial membrane inflammation in RA, resulting from the immune activation and infiltration of leucocyte. Li and colleagues examined the expression of circRNAs in synovial tissues and screened out circ-0001859 as a critical RA-related circRNA [119]. Their further studies indicated that circ-0001859 could up-regulate ATF2 via sponging miR-204/211 in synovial sarcoma cells (SW982 cells). Silencing circ_0001859 reduced the hyper inflammatory activity in the synovial tissue and alleviates the pathogenesis of RA ( Figure 5C). Circ-0088036 was found to be aberrantly upregulated in fibroblast-like synoviocytes (FLSs) in RA [120]. Circ-0088036 could promote the proliferation and migration of RA-FLSs via miR-140-3p/SIRT1 axis, subsequently promoting RA progression ( Figure 5C).
The studies described above highlight the effects of circRNAs on RA, but a great deal remains to be understood. Future studies are required to further understand the underlying mechanisms of regulation of RA progression by circRNAs.

Conclusions and future perspective
CircRNA is a class of noncoding RNA molecules with a closed loop structure formed by covalent bonds, which can protect them from degradation by most RNases [121]. Emerging studies have exploited highly-accurate circRNA biomarkers in human body fluids for diagnosis and prognosis of some diseases, such as cancer and cardiovascular diseases [122,123]. In recent years, studies have also revealed the potential value of circRNAs in clinical treatment of various diseases, including skeletal and chondral disorders. In this review, we have reviewed the regulatory role of circRNA in skeletal and chondral disorders and summarized findings of several circRNAs that play critical roles in the process of osteoarthritis, ONFH, osteoporosis, or RA. These findings may provide further insight into the mechanistic details of the role of circRNAs in bone or cartilage metabolism, and highlight potential targets for the clinical treatment of orthopedic diseases. Nevertheless, the molecular functions of circRNAs in skeletal and chondral disorders still remain largely enigmatic. Their regulatory mechanism in skeletal and chondral disorders by regulating transcription, binding protein, and encoding protein or peptides still remains unknown, which is worth to be further explored in later research on skeletal and chondral disorders.
Despite recent discoveries summarized above, many challenges remain to be overcome. The functional verification of circRNAs implicated in skeletal and chondral disorders, has been mostly in vitro. In vivo studies are a greater challenge due to the lack of stable and specific delivery vehicles. Recent studies have discovered some biological vehicle that can be loaded with circRNA (or si-circRNA) for in vivo therapy, including extracellular vesicles (EVs) and adeno-associated viral (AAV) vectors. EVs are a heterogeneous group of lipid bilayer-enclosed nanosized vesicles releasing from various types of cells [124]. Many studies have highlighted the application of EVs in transporting circRNA to specific tissues by modification [125,126]. AAV is a non-pathogenic member of the Parvovirus family which can deliver circRNA producing transgenes [127]. Studies have also revealed the potential value of recombinant AAV in targeting therapy via genetic modification [128]. These advances may shed new lights on the application of circRNA in the treatment of skeletal and chondral disorders. We believe that an in-depth understanding and correct application of circRNAs in clinical practice will make a giant progress in the treatment of skeletal and chondral disorders in the near future.