MiR-501-3p Inhibits C2C12 Proliferation Promotes C2C12 Differentiation By Targeting FOS

Skeletal muscle is essential for the exercise of important biological functions such as exercise,posture support,respiration and heat production.Skeletal muscle is the largest organ of the human body(by mass),accounting for about 40% of the mass of healthy individuals.Skeletal muscle fibers contain sarcomere,and their uniform contraction produces the power needed for the body's daily life.Satellite cells are in a static state under normal conditions,and the body is activated to become myoblasts after being damaged.It plays an important role in the growth and repair of skeletal muscle and damage repair.Therefore,the study of skeletal muscle satellite cells has important practical significance.C2C12 cells are mouse myoblast cell lines that can be widely used to study the process of myogenic differentiation.mi RNAs are short-chain,non-coding RNAs that play important roles by regulating the expression of their target genes.Studies have shown that mi R-501-3p plays an important role in diseases such as cancer,and a large number of studies have shown that mi R-501-3p can regulate cell proliferation,differentiation and migration.However,little is known about the role of mi R-501-3p in the proliferation and differentiation of C2C12.In this study,we overexpressed and interfered with mi R-501-3p,and studied its effect on proliferation,differentiation and migration of C2C12 cells,using bioinformatics methods to predict its target genes,and verifying the targeting relationship to construct a target gene overexpression vector in C2C12.The cells overexpress and interfere with the target gene,study the function of the target gene,and perform functional recovery verification in C2C12 cells.The main findings are as follows: 1.Effect of mi R-501-3p on proliferation of C2C12 cellsAfter 24 h of overexpression and interference with mi R-501-3p,the proliferation of C2C12 cells was detected by Ed U method and flow cytometry.The results showed that mi R-501-3p could inhibit the proliferation of C2C12 cells.q RT-PCR and Western blot showed that mi R-501-3p inhibited CCNA expression and up-regulated CCNB expression at m RNA and protein levels.In conclusion,mi R-501-3p can inhibit the expression of CCNB by inhibiting the expression of CCNA(P<0.01)and inhibit the proliferation of C2C12 cells.2.Effect of mi R-501-3p on the differentiation of C2C12 cellsAfter overexpressing and interfering with mi R-501-3p,C2C12 cells were induced to differentiate after about 90% confluence.Myosin immunofluorescence was performed at 3 and 5 days after differentiation,and Myo G and Myosin were detected by q RT-PCR and Western blot.The level of expression.The results showed that mi R-501-3p promoted the differentiation of C2C12 cells by promoting the relative m RNA and protein levels of Myo G and Myosin(P < 0.001).3.Effect of mi R-501-3p on migration of C2C12 cellsC2C12 cell migration was detected by scratch test 24 h after overexpression and interference with mi R-501-3p,respectively.The results showed that mi R-501-3p did not affect the migration of C2C12.4.mi R-501-3p target gene prediction and verificationBioinformatics methods were used to predict target genes,and the targeting relationship was verified by dual luciferase reporter system,q RT-PCR and Western blot.The results show that mi R-501-3p can directly target FOS.5.Effect of FOS on proliferation,differentiation and related genes of C2C12 cellsThe FOS overexpression and interference vectors pc DNA3.1-FOS and si-FOS were successfully constructed and successfully overexpressed and interfered with FOS on C2C12 cells by Ed U method,flow cytometry and Myosin immunofluorescence assay.The experimental results show that FOS promotes the proliferation and inhibits the differentiation of C2C12 cells.The results of q RT-PCR and Western blot showed that FOS promoted the expression of CCNA,inhibited the expression of CCNB,inhibited the relative m RNA and protein levels of Myo G and Myosin,and promoted the proliferation of C2C12 cells.6.mi R-501-3p regulates proliferation,differentiation and related gene expression of C2C12 cells by targeting FOSThrough functional recovery experiments(Ed U method,Myosin immunofluorescence assay),we found that co-transformation of mi R-501-3p and pc DNA3.1 inhibited the proliferation of C2C12 cells and promoted the differentiation of C2C12 cells,and this effect was co-transformed into mi R-501-3p.Reversal with pc DNA3.1-FOS;co-transfection of NC and pc DNA3.1-FOS promoted the proliferation of C2C12 cells and inhibited the differentiation of C2C12 cells.Consistent results were also obtained at the protein level.7.Myo D-mi R-501-3p-FOS-Mdfi-Myo D forms a feedback loopThrough dual luciferase reporter system and Ch IP validation,it was found that FOS inhibited Mdfi expression,Myo D increased mi R-501-3p level,and finally Myo D-mi R-501-3p-FOS-Mdfi-Myo D formed a feedback loop to regulate C2C12 myogenic differentiation.In conclusion,our results shown mi R-501-3p inhibits the C2C12 proliferation and promotes the C2C12 differentiation by inhibiting its target gene FOS expression.Our results also indicate beside the Myo D and FOS feedback loop there is also a regulatory loop formed by Myo D-mi R-501-3p-FOS-MDFI to regulate myoblast development.Our results expand our understanding of muscle myogenic development mechanism in which mi RNA and gene participate in control skeletal muscle development.