Overexpression of miRNA-133a on the in vitro proliferation and differentiation of L6 myoblasts

Objective To construct recombinant lentiviral vector of micro RNA-133a and observe the proliferation,differentiation and expression of transcription factor MEF2A of L6 myoblasts transfected with the vector system.Methods Recombinant lentiviral vector containing micro RNA-133a gene was constructed and transfected into L6 myoblasts.The expression of micro RNA-133a gene was detected by real-time PCR (Taqman probe).The effect of micro RNA-133a overexpression on L6 myoblast proliferation was quantified using cell counting kit (CCK-8).Its effect on cell differentiation was detected by inverted fluorescence microscope.Western blot assay was used to detect the expression level of transcription factor MEF2A in these cells.Results The successful construction of micro RNA-133a recombinant lentiviral vector was confirmed by plasmid enzyme digestion and DNA sequencing.Compared with the control group,relative expression of micro RNA-133a gene in L6 myoblasts was significantly increased (P ＜ 0.01) 24h after the vector transfection.L6 cell proliferation was increased significantly (P ＜ 0.01),while its differentiation was effectively inhibited.The expression level of MEF2A was significantly reduced (P ＜ 0.01).Conclusion Micro RNA-133a recombinant lentiviral vector can successfully transfect L6 myoblasts causing the cells to overexpress micro RNA-133a.This overexpression promotes L6 myoblast proliferation and inhibits its differentiation in an in vitro cell culture system. Key words: Cell differentiation;  Cell proliferation;  Lentiviral vector;  L6 myoblasts;  Micro RNA-133a;  MEF2A