Abstract: 【Objective】This experiment was designed to investigate whether DNA methylation of adipogenic promoters (peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα)) and myogenic promoter (myogenin) was involved in C2C12 myoblasts intracellular lipid accumulation.【Method】C2C12 cells were treated, respectively, by 2% horse serum and an adipogenesis cocktail which typically contains 3-isobutyl-methylxanthine, dexamethasone and insulin (MDI). In order to observe the formation of myotubes, cells were incubated for 0 d, 1 d, 3 d, and 5 d by 2% horse serum and then were collected to perform Giemsa staining. To confirm the lipid accumulation, cells treated by adipogenesis program for 0 d, 2 d, 4 d, 6 d and 10 d were fixed to conduct Oil Red O staining. Genome DNA and total RNA were extracted from C2C12 cells during the myogenesis differentiation and intracellular lipid accumulation. Real-time quantitative reverse transcription PCR was used to detect the mRNA levels of myogenic and adipogenic related genes. The bisulfite sequencing was adopted to explore the sequential DNA methylation changes in PPARγ, C/EBPα and myogenin promoter regions, and the relationship between DNA methylation and gene expression was analyzed.【Result】After horse serum treatment, the morphology of multinucleated myotubes and up-regulation of myogenic genes were displayed in C2C12 cells, but the adipocyte-specific genes showed no significant changes. The adipogenesis cocktail induced the intracellular lipid accumulation during automatical differentiation into myotubes of C2C12 cells, and both the myogenic and adipigenic genes were up-regulated. The bisulfite sequencing showed that the methylation level of PPARγ gene in undifferentiated myoblasts was 61%, and progressively demethylated upon adipogenic differentiation with methylation levels of 49%, 39% and 42% on d 2, 4 and 6, which was accompanied by an increase of mRNA level of PPARγ. But during the horse serum treatment, there were no significant changes in PPARγ methylation levels (56% and 48% on d 3 and 5) and mRNA levels. In the case of myogenin, it showed decreased methylation levels after horse serum inducement with methylation levels of 49%, 42%, 35% and 34% on d 0, 1, 3 and 5, respectively, while the expression of myogenin increased rapidly. During the adipogenic inducement, myogenin gene also demethylated (49%, 37%, 41% and 38% on d 0, 2, 4 and 6), but the degree was weaker than that in horse serum treatment, which agreed with the reduced up-regulation of myogenin mRNA level. C/EBPα showed hypomethylated status with only 1.6% in undifferentiated C2C12 cells and maintained the hypomethylated status regardless the myogenesis or lipid accumulation, so the methylation of C/EBPα promoter seemed unrelated to its transcription.【Conclusion】The dynamics of DNA methylation of PPARγ and myogenin promoter are closely correlated with their gene expression, which indicates that DNA methylation may play a role in regulating the myogenesis and lipid accumulation in C2C12 cells.

Key words: C2C12 cells , DNA methylation , myogenesis differentiation , lipid accumulation