Study On The Effects Of MicroRNA-204-5p On Proliferation And Differentiation Of C2C12 Myoblast

microRNAs(miRNAs),a class of conserved small non-coding RNAs,which can induce m RNA degradation or translation inhibition by complementary binding with the 3'UTRs of their target m RNAs.The highly complicated process of skeletal muscle development is orchestrated by a large number of regulatory factors,such as miRNA.High-throughput sequencing results found that the expression levels of miR-204-5p in porcine skeletal muscle gradually decreased with aging.However,there are few reports on how miR-204-5p regulats skeletal muscle development.In this study,C2C12 cell line was used as the research model,miR-204-5p mimics and inhibitors were transfected into C2C12 to explore the mechanism of miR-204-5p on the proliferation and differentiation of myoblasts.Further,the dual luciferase reporter system and small interfering RNA technology were used to verify whether miR-204-5p regulates myogenesis related differentiation and muscle fiber transformation through target genes.The results are as follows:(1)The expression level of miR-204-5p in murine muscle gradually decreased with the process of aging.Concurrently,the expression of miR-204-5p was demonstrated for a gradual decrease in C2C12 myoblasts during proliferation and differentiation.(2)By CCK-8 and Ed U kits of cell proliferation analysis and RT-q PCR technology,miR-204-5p was proved to significantly inhibit the cells proliferation,and the rate of Ed U positive cells and the expression of cell circle related genes were decreased significantly(p<0.01).(3)During C2C12 cell differentiation stage,through the bright-field microscopy observation and myosin heavy chain(My HC)staining,we found that miR-204-5p significantly inhibited myotube formation,and the proportion of My HC positive cells was significantly reduced(p<0.01).By RT-q PCR technique,we found that miR-204-5p significantly inhibited the expression of the myogenic differentiation marker gene(p<0.01).(4)miR-204-5p could significantly dicrease the synthesis of all types of My HC,and the reduction of My HCI was remarkable(p<0.01).Immunofluorescence staining of My HCI showed that miR-204-5p significantly inhibited the genesis of slow-twitch myofibers(p<0.01).(5)Mitochondrial staining and relative mitochondrial DNA content analysis showed that miR-204-5p significantly inhibited the formation of mitochondrial(p<0.05).(6)Dual luciferase report system verified that MEF2 C and ERR? are bona fide targets of miR-204-5p.By interfering MEF2 C or ERR? expression,miR-204-5p significantly inhibited the formation of myotube and slow-twitch fibers(p<0.01).Meanwhile,co-transfection of either si-MEF2 C or si-ERR? with miR-204-5p mimics resulted in a more severe attenuation of the formation of myotube and slow-twitch fibers(p<0.01).These results suggest that miR-204-5p could negatively regulate the proliferation and differentiation of C2C12 cells and affect the transformation of muscle fiber types and mitochondrial formation.miR-204-5p directly targets on MEF2 C and ERR? in the regulation of myogenesis related differentiation and muscle fiber transformation.Our findings imply that miR-204-5p is a potential regulator that could influence myogenesis and provide basic data for the study of epigenetic regulation of muscle development.