METTL3 Regulates The Mechanism Of Muscle Differentiation Through Wnt Signaling Pathway

Background: Mammalian skeletal muscle has the ability of self-repair after trauma,and the differentiation and regeneration of satellite cells is the key.m6A modification is the most abundant internal modification in mammalian mRNA and plays an important role in skeletal muscle differentiation.Studies have shown that skeletal muscle formation is strictly regulated by Wnt signaling pathway.There are a lot of literatures that show that m6A can regulate the progression of various cancers through Wnt signaling pathway.However,whether m6A can regulate muscle differentiation through Wnt signaling pathway is still unclear.The aim of this study was to explore the potential molecular mechanism of m6A modification in regulating muscle differentiation,and to provide new ideas for clinical treatment of muscle atrophy.Methods: RT-qPCR and Western-Blot were used to detect the expression of METTL3 during C2C12 cell differentiation,and RT-qPCR and Western-Blot were used to detect the expression of METTL3 during the repair of skeletal muscle injury.The effects of METLT3 on the proliferation of C2C12 cells were detected by Ed U assay,and the effects of METTL3 on the differentiation of C2C12 cells were detected by RT-qPCR,Western-Blot and IF techniques.The correlation between METTL3 and Wnt signaling pathway was investigated by adding CHIR99021 and 1-AKP to investigate the relationship between METTL3 and Wnt signaling pathway.The effect of METTL3 on the level of m6A in C2C12 cells was detected by Dot-Blot and total m6A level in C2C12 cells,and the effect of METTL3 on m6A level in C2C12 cells was detected by Me RIP-qPCR.RT-qPCR and Western-Blot were used to identify the target gene and the identified protein,RT-qPCR and Western-Blot were used to identify the active site of METTL3 RT-qPCR and Western-Blot demonstrated that METTL3 regulates muscle differentiation by regulating the expression of LEF1.Results: The expression of METTL3 was up-regulated during the differentiation of C2C12 cells and the repair of skeletal muscle injury,which showed that METTL3 played an important role in the differentiation of skeletal muscle.Overexpression of METTL3 can inhibit the proliferation of C2C12 cells and promote the differentiation of myoblasts.There is a potential correlation between METTL3 and Wnt signaling pathway during C2C12 cell differentiation.METTL3 can increase the level of m6A in C2C12 cells,METTL3 can affect the m6A level of key factors in Wnt signaling pathway,and METTL3 can affect the level of mRNA and protein of LEF1 and depend on its m6A active site.YTHDF1 is the recognition protein in the process of METTL3 modified LEF1.METTL3 regulates muscle differentiation by regulating the expression of LEF1.Conclusion: METTL3 promotes myoblast differentiation and repair of skeletal muscle injury through m6A modification of LEF1 in Wnt signaling pathway.