FTO m6A demethylase positively regulates circZCCHC14/miR-181a/GREM1 and BMP2 axis in peripheral blood-derived mesenchymal stem cells (PBMSCs) chondrogenic differentiation of Diannan small ear pigs

Abstract

Background Circular RNAs (circRNAs) are a class of noncoding RNAs that are involved in chondrogenic differentiation, and N6-methyladenosine (m⁶A) broadly exists in circRNAs. Materials and methods A joint injury model was constructed on Diannan small-ear (DSE) pigs. Transfections were constructed using Lipofectamine 2000. Real-time quantitative PCR (qPCR), Methylated RNA immunoprecipitation qPCR (MeRIP-qPCR), and western blotting analyses were performed. Alcian blue staining tested the chondrogenic differentiation ability. The potential m⁶A methylation modification enzymes and sites of circZCCHC14 were predicted in m6Avar and SRAMP databases. RNA pull-down and RIP assays were conducted to determine the interaction between circZCCHC14 and FTO. Results CircZCCHC14 expression and the m⁶A methylation level were increased in joint injury DSE pigs. m⁶A methylation and circZCCCHC14 expression levels were decreased during the process of cartilage differentiation. FTO was decreased, circZCCHC14 and m⁶A methylation level were increased under inflammatory conditions. FTO is one essential m⁶A demethylase enzyme of circZCCHC14. m⁶A demethylase enzyme FTO regulated the expression levels of circZCCHC14. m⁶A demethylase enzyme FTO positively regulated the expression of miR-181a. FTO m⁶A demethylase positively regulates chondrogenic differentiation through the circZCCHC14/miR-181a/GREM1 axis. Conclusion Our data showed the physiological significance of FTO m⁶A demethylase in regulating axis circZCCHC14/miR-181a/GREM1 and BMP2, providing a potentially effective therapeutic target for the treatment of joint injury or Osteoarthritis (OA).