Involvement Of MiR-20a/106b In The Regulation Of Myoblast Proliferation And Differentiation

Increasing evidences have suggested that microRNAs play an important role in regulating skeletal muscle development. Microarrays have found the dynamic expression pattern of miR-20a and106b during myogenesis, but their involvment in myoblast proliferation and differentiation remains elusive. Using bioinformatic analysis we identified a putative miR-20a target site in the3’-UTR of Myf5, as well as a miR-106b potential target site in Akt3. Considering both Myf5and Akt3have estalished role in myogenesis, we set to investigate the regulation of miR-20a/106b on Myf5/Akt3during C2C12myoblast proliferation and differentiation..The results are as follows:1、Luciferase reporter assay in BHK-21cells revealed that miR-20a and miR-106b mimics significantly reduce the luciferase activity of Myf5-3’UTR construct. No such effect was observed when cells were transfected with either miR-20a and miR-106b inhibitors or construct harboring mutant3’UTR of Myf5. Furthermore, overexpression of miR-20a mimics in C2C12cells markedly reduced the endogenous MYF5protein expression, whereas co-transfection of miR-20a inhibitors could counteract this effect.2、Luciferase reporter assay in BHK-21cells revealed that miR-106b mimics significantly reduce the luciferase activity of Akt3-3’UTR construct. No such effect was observed when cells were transfected with either miR-106b inhibitors or construct harboring mutant3’UTR of Akt3.3、Flow cytometry revealed that C2C12myoblasts transfected with either miR-20a or miR-106b mimics exhibit growth retardation, demonstrated by an accumulation of cell in the G0/G1phase of cell cycle.Taken together, our data suggested that miR-20a can regulate the expression of its target gene Myf5and miR-106can regulate the expression of its target gene Myf5and Akt3during myogenesis, which in turn affects the proliferation and differentation of myoblasts.