| The growth and development of skeletal muscle is a highly systematic and complex biological process regulated by a variety of genes.More and more studies have shown that mi RNAs are involved in the regulation of differentiation,proliferation and apoptosis of skeletal muscle satellite cells(SMSCs).MEF2 C gene is not only a marker gene in myogenic differentiation of myoblasts,but also a potential target gene of mi R-194-5p.A large number of studies have reported that mi R-194-5p is involved in the regulation of cancer cell proliferation,but its involvement in regulating the proliferation of skeletal muscle cells has not been reported.In this experiment,rabbit skeletal muscle satellite cells and C2C12 were used as research objects.Up-regulation or down-regulation of mi R-194-5p was used to study the effects of mi R-194-5p on proliferation and differentiation of rabbit SMSCs and apoptosis of C2C12.Results are as follows:1.The expression of mi R-194-5p in heart,liver,spleen,stomach,kidney and muscle tissues of New Zealand rabbits at 84 days old was measured.The results showed that the expression levels of mi R-194-5p in muscle was the highest and significantly higher than other tissues(p<0.05).This indicated that mi R-194-5p expressed specially in rabbit muscle.2.Using online software,those target genes of mi R-194-5p were clustered using GO and KEGG analysis.The results showed that these target genes are involved in multiple signaling pathways regulating the growth and development of the body.3.In the SMSCs proliferation assay,CCK-8 results showed that the number of viable ells in the mi R-194-5p mimic group was significantly lower than the NC group(p<0.05);The results of Ed U showed that the percentage of Ed U positive cells in the inhibitor mi R-194-5p group was significantly higher than that in the inhibitor NC group(p<0.01),it indicated that mi R-194-5p could impede the proliferation of rabbit SMSCs.4.MEF2 C was successfully confirmed as a target gene of mi R-194-5p by dual luciferase reporter system.The expression level of MEF2 C gene in mi R-194-5p mimic group was significantly lower than the NC group on the 1st day of differentiation of SMSCs(p<0.05).5.In the SMSCs differentiation assay,the expression level of myoblast differentiation gene Myo G in the mi R-194-5p mimic group was lower(significantly)than that of the NC group on the 3rd,and 7th day of differentiation(p<0.05,p<0.01).It indicated that mi R-194-5p suppressed the expression of Myo G.6.Apoptosis of C2C12 was induced by H2O2,and the number of apoptotic cells in the mi R-194-5p mimic,NC,inhibitor and in NC groups was detected by flow cytometry.The results showed that compared with the control group,the apoptosis rate of mi R-194-5p group was extremely significant higher(p<0.01).As for Western blotting,the expression of anti-apoptotic protein Bcl-2 in the mi R-194-5p up-regulated group was significantly lower than that in the control group,and the expression of the pro-apoptotic protein caspase-3 in the inhibition group was significantly lower than that in the control group(p<0.05).It was suggested that mi R-194-5p promoted H2O2-induced apoptosis of C2C12 cells.All above result showed that mi R-194-5p expressed specially in rabbit muscle and inhibited the proliferation of rabbits' SMSCs.mi R-194-5p targeted MEF2 C and inhibited the expression of Myo G;mi R-194-5p promotes H2O2-induced apoptosis of C2C12. |
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