The Effect Of MiR-204-5p On The Myogenic Differentiation Of C2C12

The carcass lean percentage in pigs is substantially detennined by the number of skeletal muscle fibers.The growth and development of pig skeletal muscle is a complex biological process regulated by many transcription factors.It is well-known that microRNAs(miRNAs)exert significant roles in skeletal myogenesis.The previous studies have shown that miR-204-5p was highly expressed in skeletal muscle at prenatal stage,but barely expressed in adult skeletal muscle,yet there is no report about the role of miR-204-5p and its molecular mechanism in skeletal myogenesis.The growth and differentiation activity of C2C12 myoblasts was investigated,then the mimics and inhibitors of miR-204-5p were transfected.The marker protein Myhl of myotubes in induction(ID)group,mimics(MI)group and inhibitors(IN)group was stained by immunofluorescent staining.Furthermore,genome-wide expression profiles in ID,MI and IN group were examined by using RNA-seq technologies.The potential target genes of miR-204-5p were screened out through the differentially expressed transcription analysis,and were further validated by qPCR.The results of immunofluorescent staining showed that the expression level of Myhl in ID and IN group were obviously higher than that in MI group;The myoblasts fusion rate in IN group was higher,and myotubes arranged more regular than that of ID group.The result suggested that miR-204-5p could significantly inhibit the myoblasts fusion rate,thus reduce skeletal myogenesis.A total of 1750379,18986284 and 20444580 clean read pairs were obtained from the ID,MI and IN group,respectively.After alignment against the mouse reference genome(GRCm38.85)by STAR(v2.5.2a)software,the number of the uniquely mapped reads was 15737764(90.87%)17197729(90.58%),and 18616465(91.06%)in ID,MI,and IN group,respectively.We then normalized the raw read counts of each gene by TMM to identify significantly differentially expressed genes(DEGs).The result showed that there were totally 78 DEGs in ID and MI group by Deseq(FDR<0.5),among which 34 genes were more highly expressed in ID group,but the remaining 44 genes were lowly expressed.The number of genes highly expressed in ID vs.IN group was 34(FDR<0.5),the abundance of other 67 genes were lower.In ID vs.IN group,the number of genes differentially expressed was 39,among which 13 genes were more highly expressed in MI group.Through the comparative analysis of the differentially expressed genes and the corresponding counts per million values of each gene.13 potential target genes of miR-204-5p were selected,including Lrp4,Clqtnf3,Myh3.Olfml2b,Pdlim3,Myl4,Myhl,Myh4,Sema3c.Actcl,Aspn,Ogn and Mef2c.The six genes Lrp4,Mef2c.Pdlim3,Clqtnf3,Sema3c and Ogn were then thought to be plausible target genes of miR-204-5p using miRNA online predicted software RNA hybrid 2.2 and confirmed by qPCR.Taken together,these results demonstrated that miR-204-5p might inhibits the myogenic differentiation of C2C12 by targeting Lrp4,Mef2c,Pdlim3,Clqtnf3,Sema3c and Ogn,thus suppressed the formation of myotubes through inhibiting the fusion of myoblasts,...