Study On The Mechanism Of MiR-532-5p Regulating The Differentiation Of C2C12 Myoblasts

As an important tissue of the human body,skeletal muscle plays a key role in maintaining body movement and regulating energy metabolism.Recent studies have found that miRNA plays an important role in the differentiation of skeletal muscle cells.miR-532-5p plays a role in regulating cardiomyocyte apoptosis and osteoblast differentiation,but its function and mechanism in skeletal muscle differentiation are not clear.In this study,mouse myoblast C2C12 was induced to differentiate into myoblasts,and then the effect of miR-532-5p on myoblast differentiation of C2C12cells was investigated by overexpression of miR-532-5p and knockdown of target genes,and the mechanism of miR-532-5p on myoblast differentiation was also discussed.1.Optimization of myogenic differentiation conditions of C2C12 cellsIn order to optimize the myogenic conditions of C2C12 cells,different numbers of C2C12 cells were inoculated into the culture medium containing different concentrations of horse serum.The results showed that inoculating 5.00×10~4 C2C12cells into the culture medium containing 2%horse serum is the best myogenic differentiation condition.2.Expression of miR-532-5p in C2C12 cells during myogenic differentiation and in different tissues of mouseThe cell samples of 3 days,5 days and 7 days of differentiation were collected and detected by q RT-PCR.The results showed that the expression of miR-532-5p decreased gradually with the increase of C2C12 cell differentiation time,and the results of q RT-PCR detection showed that the expression of miR-532-5p was the highest in mouse fat,followed by heart and muscle.3.Effect of miR-532-5p overexpression on myogenic differentiation of C2C12cellsmiR-532-5p mimics was transfected into C2C12 cells,and then the myotubes differentiated for 3 days,5 days and 7 days were stained with Giemsa and Immunofluorescence staining.The length,diameter and fusion rate of myotubes were calculated.The results showed that the muscle tube length and fusion rate decreased significantly.q RT-PCR and WB were used to detect the expression of myogenic marker genes at 3 days,5 days and 7 days of differentiation.The results showed that the expressions of myogenic marker genes Myod,Myog and Myf5 were significantly down-regulated at transcriptional and protein levels.It is suggested that the overexpression of miR-532-5p inhibits the myogenic differentiation of C2C12 cells.4.Screening and verification of target genesIn order to explore the mechanism of miR-532-5p regulating myoblast differentiation,six candidate target genes of miR-532-5p were screened through the target gene prediction website and the score of each gene.Double luciferase verification test showed that Frs2 was the target gene of miR-532-5p.q RT-PCR and WB were used to detect the changes of Frs2 expression after overexpression of miR-532-5p.The results showed that the transcriptional and protein levels of Frs2were significantly decreased.It was further confirmed that Frs2 was the target gene of miR-532-5p.5.Effect of Frs2 knockdown vector on myogenic differentiation of C2C12 cellsqRT-PCR was used to detect the expression of Frs2 in C2C12 cells at 3 days,5days and 7 days,as well as in different tissues of mouse.The results showed that Frs2showed an upward trend during myogenic differentiation,its expression was the lowest in mouse fat,followed by stomach and muscle.Frs2 was knocked down and transferred into C2C12 cells,the myotubes induced for 3 days,5 days and 7 days were stained with Giemsa and Immunofluorescence staining.The length,diameter and fusion rate of myotubes were calculated.The results showed that the muscle tube length and fusion rate decreased significantly.Then the expression of myogenic marker genes was detected by q RT-PCR and WB.The results showed that the transcriptional and protein levels of Myod,Myog and Myf5 decreased significantly.The above results indicate that the knockout of Frs2 inhibits the myogenic differentiation of C2C12 cells,which is basically consistent with the overexpression of miR-532-5p.To sum up,miR-532-5p regulates the myogenic differentiation of C2C12 cells by regulating the expression of Myod,Myog and Myf5 by regulating the target gene Frs2.These results provide an experimental basis for exploring the mechanism of muscle differentiation,diagnosis and treatment of diseases.