Objective To investigate the effect and mechanism of Bmal1 overexpression on C2C12 myoblasts proliferation and differentiation. Methods Bmal1 was overexpressed in C2C12 myoblasts by lentivirus-mediated stable transfection. The expression levels of mRNA and protein of some myogenic regulatory factors were determined by RT-qPCR or Western blot assay. Transcriptome sequence analysis and its related experiments were used to explore the expression of Wnt signaling pathway and cell cycle regulators in C2C12 myoblasts after overexpression of Bmal1 during the differentiation process. Subsequently, Wnt signaling pathway was blocked by inhibitor XAV939. The effects of XAV939 on C2C12 myoblasts after overexpression of Bmal1 were observed using corresponding experiments. Results Overexpression of Bmal1 promoted the proliferation of myoblasts cultured in growth medium. The S-phase fraction of total cells (SPF) and Proliferation Index (PI) of myoblasts increased. Compared with the blank control group and negative control group, the ratio of EdU positive cells and nuclear translocation of β-catenin were increased in C2C12 myoblasts after overexpression of Bmal1. Overexpression of Bmal1 significantly promoted myogenic differentiation and myotube fusion. The mRNA and protein expression levels of MyoD, myogenin, and Myh3 were up-regulated in a time-dependent manner in C2C12 myoblasts after overexpression of Bmal1. Cluster analysis and related experiments supported that the expression of the relative factors of Wnt signaling pathway and cell cycle was up-regulated during the proliferation and differentiation of Bmal1-overexpressed C2C12 myoblasts. XAV939(5μmol/L) treatment inhibited the Bmal1 overexpression-induced the up-regulated expression of Tcf1 and c-Jun. Continuous XAV939 treatment suppressed myotube fusion and down-regulated the expression of MyoD, myogenin, and Myh3 in Bmal1-overexpressed C2C12 myoblasts. Conclusions Bmal1 may serve as a potential target for skeletal muscle injury repair. Overexpression of Bmal1 enhances myoblasts proliferation and differentiation by enhancing Wnt signaling pathway.
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