Long non-coding RNA CIR inhibits chondrogenic differentiation of mesenchymal stem cells by epigenetically suppressing ATOH8 via methyltransferase EZH2

Abstract

Background: Osteoarthritis (OA) is the most common articular disorder, leading to joint malfunction and disability. Although the incidence of OA is increasing globally, the treatment of OA is very limited. LncRNA CIR has been implicated in OA through unclear mechanisms. Here, we investigated the role of lncRNA CIR in chondrogenic differentiation. Methods: Human umbilical-cord-derived mesenchymal stem cells (hUC-MSCs) were obtained from human umbilical cords. Flow cytometry was used to analyze the surface markers of hUC-MSCs. Various culture conditions and corresponding staining assays were employed to assess the differentiation abilities of hUC-MSC. qRT-PCR, western blot, and immunostaining were used to measure expression levels of related genes and proteins such as lncRNA CIR, ATOH8, EZH2, and H3K27me3. RNA immunoprecipitation assay, biotin pull-down, and chromatin immunoprecipitaion assay were performed to analyze the interactions of lncRNA CIR, EZH2, H3K27me3 and ATOH8 promoter. Results: hUC-MSCs exhibited MSCs features and could differentiate into chondrocytes under specific conditions. LncRNA CIR was downregulated while ATOH8 was upregulated during the chondrogenic differentiation of hUC-MSCs. Knockdown lncRNA CIR or overexpression of ATOH8 promoted chondrogenic differentiation. Further, lncRNA CIR bound to EZH2 and repressed ATOH8 expression via EZH2-mediated H3K27me3, which promotes the methylation of ATOH8. Inhibition of ATOH8 reversed the effects of knockdown lncRNA CIR on chondrogenic differentiation. Conclusion: LncRNA CIR suppresses chondrogenic differentiation of hUC-MSCs. Mechanistically, lncRNA CIR could inhibit ATOH8 expression that functions to promote chondrogenic differentiation through EZH2-mediated epigenetic modifications.