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SETD3 is a member of SET-domain containing methyltransferase family, which plays critical roles in various biological events. It has been shown that SETD3 could regulate the transcription of myogenic regulatory genes in C2C12 differentiation and promote myoblast determination. However, how SETD3 is regulated during myoblast differentiation is still unknown. Here, we report that two important microRNAs (miRNAs) could repress  SETD3  and negatively contribute to myoblast differentiation. Using microRNA (miRNA) prediction engines, we identify and characterize miR-15b and miR-322 as the primary miRNAs that repress the expression of  SETD3  through directly targeting the 3’-untranslated region of  SETD3  gene. Functionally, overexpression of miR-15b or miR-322 leads to the repression of endogenous  SETD3  expression and the inhibition of myoblast differentiation, whereas inhibition of miR-15b or miR-322 derepresses endogenous  SETD3  expression and facilitates myoblast differentiation. In addition, knockdown  SETD3  in miR-15b or miR-322 repressed myoblasts is able to rescue the facilitated differentiation phenotype. More interestingly, we revealed that transcription factor E2F1 or FAM3B positively or negatively regulates miR-15b or miR-322 expression, respectively, during muscle cell differentiation, which in turn affects  SETD3  expression. Therefore, our results establish two parallel cascade regulatory pathways, in which transcription factors regulate microRNAs fates, thereby controlling  SETD3  expression and eventually determining skeletal muscle differentiation.

MiR-15b and miR-322 inhibit SETD3 expression to repress muscle cell differentiation