Objectives: Aberrations in exosomal circular RNA (circRNA) expression have been identified in various human diseases. In this study, we investigated whether exosomal circRNAs could act as competing endogenous RNAs (ceRNAs) to regulate the pathological process of osteoarthritis (OA). This study aimed to elucidate the specific MSC-derived exosomal circRNAs responsible for MSC-mediated chondrogenic differentiation using human bone marrow-derived MSCs (hMSCs) and a destabilization of the medial meniscus (DMM) mouse model of OA. Methods: Exosomal circRNA deep sequencing was performed to evaluate the expression of circRNAs in human bone marrow-derived MSCs (hMSCs) induced to undergo chondrogenesis from day 0 to day 21. The regulatory and functional roles of exosomal circRNA_0001236 were examined on day 21 after inducing chondrogenesis in hMSCs and were validated in vitro and in vivo. The downstream target of circRNA_0001236 was also explored in vitro and in vivo using bioinformatics analyses. A luciferase reporter assay was used to evaluate the interaction between circRNA_0001236 and miR-3677-3p as well as the target gene sex-determining region Y-box 9 (Sox9). The function and mechanism of exosomal circRNA_0001236 in OA were explored in the DMM mouse model. Results: Upregulation of exosomal circRNA_0001236 enhanced the expression of Col2a1 and Sox9 but inhibited that of MMP13 in hMSCs induced to undergo chondrogenesis. Moreover, circRNA_0001236 acted as an miR-3677-3p sponge and functioned in human chondrocytes via targeting miR-3677-3p and Sox9. Intra-articular injection of exosomal circRNA_0001236 attenuated OA in the DMM mouse model. Conclusions: Our results reveal an important role for a novel exosomal circRNA_0001236 in chondrogenic differentiation. Overexpression of exosomal circRNA_0001236 promoted cartilage-specific gene and protein expression through the miR-3677-3p/Sox9 axis. Thus, circRNA_0001236-overexpressing exosomes may alleviate cartilage degradation, suppressing OA progression and enhancing cartilage repair. Our findings provide a potentially effective therapeutic strategy for treating OA.

MicroRNAs play critical roles in the pathogenesis of osteoarthritis, the most common chronic degenerative joint disease. Exosomes derived from miR‐95‐5p‐overexpressing primary chondrocytes (AC‐miR‐95‐5p) may be effective in treating osteoarthritis. Increased expression of HDAC2/8 occurs in the tissues and chondrocyte‐secreted exosomes of patients with osteoarthritis and mediates cartilage‐specific gene expression in chondrocytes. We have been suggested that exosomes derived from AC‐miR‐95‐5p (AC‐miR‐95‐5p‐Exos) would enhance chondrogenesis and prevent the development of osteoarthritis by directly targeting HDAC2/8. Our in vitro experiments showed that miR‐95‐5p expression was significantly lower in osteoarthritic chondrocyte‐secreted exosomes than in normal cartilage. Treatment with AC‐miR‐95‐5p‐Exos promoted cartilage development and cartilage matrix expression in mesenchymal stem cells induced to undergo chondrogenesis and chondrocytes, respectively. In contrast, co‐culture with exosomes derived from chondrocytes transfected with an antisense inhibitor of miR‐95‐5p (AC‐anti‐miR‐95‐5p‐Exos) prevented chondrogenic differentiation and reduced cartilage matrix synthesis by enhancing the expression of HDAC2/8. MiR‐95‐5p suppressed the activity of reporter constructs containing the 3ʹ‐untranslated region of HDAC2/8, inhibited HDAC2/8 expression and promoted cartilage matrix expression. Our results suggest that AC‐miR‐95‐5p‐Exos regulate cartilage development and homoeostasis by directly targeting HDAC2/8. Thus, AC‐miR‐95‐5p‐Exos may act as an HDAC2/8 inhibitor and exhibit potential as a disease‐modifying osteoarthritis drug.

Background/Aims: Aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) are secreted enzymes belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family that play significant roles in the progression of osteoarthritis (OA). Here, we aimed to determine whether the expression of ADAMTS-4/5 in chondrogenesis and inflammation is regulated by microRNA-92a-3p (miR-92a-3p). Methods: MiR-92a-3p and ADAMTS-4/5 expressions were determined by quantitative polymerase chain reaction (qPCR). To investigate the repressive effect of miR-92a-3p on ADAMTS-4/5 expression, chondrogenic human mesenchymal stem cells (hMSCs) and human chondrocytes were transfected with mature miR-92a-3p or an antisense inhibitor (anti-miR-92a-3p), respectively. ADAMTS-4/5 protein production was quantified by enzyme-linked immunosorbent assay (ELISA), and miR-92a-3p involvement in IL-1β-mediated catabolic effects was examined by immunoblotting. The roles of activated MAP kinases (MAPK) and nuclear factor (NF)-κB were evaluated by using specific inhibitors. Interaction between miR-92a-3p and its putative binding site in the 3′-untranslated region (3′-UTR) of ADAMTS-4/5 mRNA was confirmed by luciferase reporter assay. Results: miR-92a-3p expression was elevated in chondrogenic hMSCs, with significantly lower expression in OA cartilage than in normal cartilage. Stimulation with IL-1β significantly reduced miR-92a-3p expression in primary human chondrocytes (PHCs). Transfection of chondrocytes with miR-92a-3p downregulated IL-1β-induced ADAMTS-4/5 expression, and the activity of a reporter construct containing the 3′-UTR of human ADAMTS-4/5 mRNA. MiR-92a-3p expression was suppressed upon IL-1β-induced activation of MAPK and NF-κB in chondrocytes. Conclusion: MiR-92a-3p is an important regulator of ADAMTS-4/5 in human chondrocytes and may contribute to the development of OA.

Osteoarthritis (OA) is an age-related cartilage-degenerating joint disease. Mitochondrial dysfunction has been reported to promote the development of OA. Poly (ADP-ribose) polymerase family member 12 (PARP12) is a key regulator of mitochondrial function, protein translation, and inflammation. However, the role of PARP12 in OA-based cartilage degradation and the underlying mechanisms are relatively unknown. Here, we first demonstrated that PARP12 inhibits mitophagy and promotes OA progression in human OA cartilage and a monosodium iodoacetate-induced rat OA model. Using mass spectrometry and co-immunoprecipitation assay, PARP12 was shown to interact with ISG15, upregulate mitofusin 1 and 2 (MFN1/2) ISGylation, which downregulated MFN1/2 ubiquitination and SUMOylation, thereby inhibiting PINK1/Parkin-dependent chondrocyte mitophagy and promoting cartilage degradation. Moreover, inflammatory cytokine-induced interferon regulatory factor 1 (IRF1) activation was required for the upregulation of PARP12 expression, and it directly bound to the PARP12 promoter to activate transcription. XAV-939 inhibited PARP12 expression and suppressed OA pathogenesis in vitro and in vivo. Clinically, PARP12 can be used to predict the severity of OA; thus, it represents a new target for the study of mitophagy and OA progression. In brief, the IRF1-mediated upregulation of PARP12 promoted cartilage degradation by inhibiting PINK1/Parkin-dependent mitophagy via ISG15-based attenuation of MFN1/2 ubiquitylation and SUMOylation. Our data provide new insights into the molecular mechanisms underlying PARP12-based regulation of mitophagy and can facilitate the development of therapeutic strategies for the treatment of OA.

Chondrocyte hypertrophy, regulated by Runt-related transcription factor 2 (RUNX2) and matrix metalloproteinase 13 (MMP13), is a crucial step in cartilage degeneration and osteoarthritis (OA) pathogenesis. We previously demonstrated that microRNA-381 (miR-381) promotes MMP13 expression during chondrogenesis and contributes to cartilage degeneration; however, the mechanism underlying this process remained unclear. In this study, we observed divergent expression of miR-381 and histone deacetylase 4 (HDAC4), an enzyme that directly inhibits RUNX2 and MMP13 expression, during late-stage chondrogenesis of ATDC5 cells, as well as in prehypertrophic and hypertrophic chondrocytes during long bone development in E16.5 mouse embryos. We therefore investigated whether this miRNA regulates HDAC4 expression during chondrogenesis. Notably, overexpression of miR-381 inhibited HDAC4 expression but promoted RUNX2 expression. Moreover, transfection of SW1353 cells with an miR-381 mimic suppressed the activity of a reporter construct containing the 3′-untranslated region (3′-UTR) of HDAC4. Conversely, treatment with a miR-381 inhibitor yielded increased HDAC4 expression and decreased RUNX2 expression. Lastly, knockdown of HDAC4 expression resulted in increased RUNX2 and MMP13 expression in SW1353 cells. Collectively, our results indicate that miR-381 epigenetically regulates MMP13 and RUNX2 expression via targeting of HDAC4, thereby suggesting the possibilities of inhibiting miR-381 to control chondrocyte hypertrophy and cartilage degeneration.