Effects Of MiR-31-5p In Proliferation And Myogenic Differentiation Of C2C12 Cell Lines

Background: mi RNAs are endogenous small non-coding RNAs, and can recognize the 3’UTR of target m RNA through complementary binding of the mi RNA, which can participate in diverse biological process such as development, differentiation and tumorigenesis. The process of skeletal muscle proliferation and differentiation can be regulated by varied factors. The key factors that control the determination and differentiation of skeletal muscle cells are myogenic regulatory factors(MRFs), are including Myo D, Myf5, Myogenin and MRF4. Myo D and Myf5 are required for the early myogenic process, whereas myogenin and MRF4 play a critical role in the terminal myogenic process. C2C12 myoblast cell line is a widely used model to study skeletal muscle myogenesis in vitro. Changing the serum conditions could induce C2C12 cells exit cell cycles and begin to fusion. Recently research of mi R-31-5p mainly focused on the regulation mechanisms of effect the biological behavior of tumors. However, the particular regulatory mechanism of mi R-31-5p in skeletal muscle development is still not so clear.Objective: To investigate the C2C12 cell proliferation activity and the relative expression of MRFS(Myo D and Myogenin) influence by mi R-31-5p in vitro. Discuss the possible mechanism of regulation according to the experimental results. Expect to provide some basis for the further research about the regulation mechanism between mi RNA and skeletal muscle development.Methods: 1. Test the expression levels of mi R-31-5p in the process of C2C12 cell myogenic differentiation by q RT-PCR; 2. Use the lipofection transfection to achieve the ‘gain of function’ or ‘loss of function’ research and test the transfection efficiency; 3. Detect the C2C12 cell proliferation activity after transfection by cell counting kit 8(CCK-8); 4. Detect the m RNA expression level changes of MRFs including Myo D and Myogenin by q RT-PCR in the C2C12 myogenic differentiation stage after the transfection.Results: 1. The expression level of mi R-31-5p decreased dynamically in C2C12 cell myogenesis(p<0.001).2. q RT-PCR results showed that mi R-31-5p mimics, inhibitor and negative control are transfected successfully. The expression of mi R-31-5p in the mimics treatment group is 42 times higher than the negative control(p<0.01), and the expression of mi R-31-5p in the inhibition treatment group is also lower than control.3. The proliferation activity of C2C12 cell significant reduced comparison with negative control group at 48 h after upregulation of mi R-31-5p(p<0.01), while inhibition of mi R-31-5p caused the cell proliferation activity obvious decreased at 24 h, 36 h and 48h(p<0.01).4. Upregulation of mi R-31-5p, the expression of Myo D and Myogenin significantly decreased than control group at the fourth day in the differentiation stage(p<0.01). And inhibition of mi R-31-5p, the expression of Myo D and Myogenin obviously increased.Conclusion:mi R-31-5p may inhibit the cell proliferation of C2C12 cell. Expression level of mi R-31-5p changed dynamically following the process of induced myogenic differentiation, and also negatively control the expression level of Myo D and Myogenin. It indicates that mi R-31-5p may affect the cell proliferation and myogenic differentiation of C2C12 cell line in vitro.