The Role And Mechanism Of MiR-125a-5p In C2C12 Cells Proliferation And Differentiation

Micro RNAs(mi RNAs) are a class of small non-coding RNAs composed of 20–25 nucleotides, which can affect m RNA stability or inhibit protein translation usually by targeting the 3’UTR region of m RNAs. Mi RNAs play an important regulatory role during the processes of myoblasts proliferation and differentiation. Currently, it has been known that mi R-133 and mi R-27 a could promote the proliferation of myoblasts. Mi R-1 and mi R-206 could promote myoblasts differentiation. However, there are still many mi RNAs of which the function and mechanism are not clear in myogenesis. In this study quantitative Real-time PCR, western blot, flow cytometry and cell immunofluorescence were used to investigate the regulatory role of mi R-125a-5p in the process of C2C12 cells proliferation and differentiation.C2C12 cell lines were used as reseach material in this experiment, oligo mimic and inhibitor were respectively transfected into the cells to overexpress or silence mi R-125a-5p. And then study the regulatory role of mi R-125a-5p during myoblasts proliferation and differentiation. Based on the regulatory mechanism of mi RNAs, bioinformatics software Targetscan were applied to predict the target genes of mi R-125a-5p and then genes related with myoblast proliferation and differentiation were selected. Then psicheck-2 dual luciferase gene reporter plasmids containing of the m RNAs 3′UTR were constructed. They were co-transfected with mi R-125a-5p mimic into HEK293 T cells and then fluorescence intensity was deteced. This study mainly got the following results:1. Mi R-125a-5p inhibits C2C12 cells proliferation.During cell proliferative phase of C2C12, the expression level of mi R-125a-5p and E2F3 displayed inverse pattern. Overexpression of mi R-125a-5p could inhibit the expression level of cell cycle protein Cyclin D1(P < 0.05), DNA replication(P < 0.05), and result in G1 phase arrest of C2C12(P < 0.05). Blocking the expression of mi R-125a-5p, showed no effect on the expression level of cell cycle protein Cyclin D1, DNA replication and cell cycle.2. Mi R-125a-5p has no significant effect on C2C12 differentiationNeither the overexpression nor interference of mi R-125a-5p could affect the expression level of myoblasts differentiation marker genes Myo D or My HC, and myoblasts differentiation index.3. E2F3 is target of mi R-125a-5pInterferencing the expression of E2F3 could inhibit the expression level of cell cycle protein Cyclin D1(P < 0.05). Through dual luciferase gene reporter assay, E2F3 is target of mi R-125a-5p.In conclusion, overexpression of mi R-125a-5p could inhibit C2C12 cells proliferation by targeting E2F3, without affecting their differentiation.