Study On The Effects Of MicroRNA-10b-5p On Proliferation,Differentiation And Migration Of C2C12 Myoblast

microRNAs(miRNAs),a class of conserved 22 nt non-coding RNAs,which is widely existing in eucaryon and can induce mRNA degradation or translation inhibition by complementary binding with the 3'UTRs of their target mRNAs.It is involved in almost all biological functions.Skeletal muscle,which accounts for about 30-40% of body weight,is an active center of metabolism and an important economic part of meat animals.However,the potential epigenetic regulation pathways of miRNAs in skeletal muscle development are not well understood.Our previous high-throughput sequencing results found that miR-10b-5p was highly expressed in porcine skeletal muscle,but the effect of miR-10b-5p on skeletal muscle development and its potential mechanism have not been reported.Therefore,in this study,C2C12 cell line was used as the research model,miR-10b-5p mimics and inhibitors were transfected into C2C12 cell to explore the mechanism of miR-10b-5p on the proliferation,differentiation and migration of myoblasts.Further,the dual luciferase reporter system and small interfering RNA technology were used to verify whether miR-10b-5p regulates myogenesis related proliferation and differentiation through target genes.The genetic regulatory pathway involving miR-10b-5p in the regulation of proliferation and differentiation of myoblasts was identified.The main results are as follows:(1)miR-10b-5p was expressed in the heart,liver,spleen,lung,kidney,brain,muscle and adipose tissue,and its expression was relatively high in the muscle tissue.At the same time,the expression level of miR-10b-5p has a certain time sequence.It was shown that the expression of miR-10b-5p steadily decreased during myoblasts proliferation(P<0.01),but significantly increased during myoblasts differentiation(P<0.01).(2)By CCK8 and EdU kits of cell proliferation analysis,miR-10b-5p was proved to markedly promote the myoblasts proliferation(P<0.01).During this process,miR-10b-5p significantly promoted the expression of cell cycle protein genes(CDK4 ? CyclinE ?CyclinD1?CyclinB)(P<0.01).(3)MyHC immunofluorescence staining showed that miR-10b-5p significantly inhibited myotube formation(P<0.01),thereby reducing myoblast differentiation.During this process,miR-10b-5p significantly inhibited the expression of the myogenic differentiation marker genes(MyHC?MyoD?MyoG?Myf5?MRF4)(P<0.01).(4)Through the cell scratch test,we found that miR-10b-5p could significantly promote the migration ability of C2C12 myoblasts(P<0.01),and significantly increased the expression of genes related to myoblast migration(Pax3?Fhl1?Actg1)(P<0.01).(5)Dual luciferase report system verified that NFAT5 and E2F7 are bona fide targets of miR-10b-5p.By interfering NFAT5 expression,miR-10b-5p significantly inhibited the formation of myotube(P<0.01)while interfering with E2F7 expression,miR-10b-5p significantly promoted the cells proliferation(P<0.01).These results suggest that miR-10-5p could positively regulate the proliferation and migration of C2C12 cells and restrain myoblasts differentiation.miR-10b-5p directly targets on E2F7 and NFAT5 in the regulation of myogenesis related proliferation and differentiation.Our findings imply that miR-10b-5p is an important epigenetic regulator in the process of myogenesis and provide basic data for the study of epigenetic regulation of muscle development.