MiR-2425 Targets RAD9A And MYOG To Regulate The Proliferation And Differentiation Of Bovine Skeletal Muscle-derived Satellite Cells

Skeletal muscle satellite cells are precursor cells of muscle tissue,which are strong plasticity and have many important biological characteristics of skeletal muscle.The activation,proliferation and differentiation of muscle satellite cells are also important mechanisms of enlargement and enlargement of skeletal muscle cells and muscle fiber transformation.The study of the regulation of muscle cell proliferation and differentiation can provide important help for muscular developmental mechanisms.MicroRNA(miRNA)is a novel non-protein expression factor for gene expression.It regulates the expression of endogenous single-stranded noncoding RNA(~ 22-nt long)at post-transcriptional level.By pairing with mRNA,miRNAs down-regulate gene expression by inhibiting translation or stimulating mRNA degradation.In some cases,they can also up-regulate the expression of the target gene.Many miRNA target gene expression for the normal development of muscle has a vital regulatory role.But the current understanding of miRNA in muscle is very limited,many miRNA regulation of muscle development details are still not very clear.Therefore,it is urgent to identify new miRNAs and find miRNAs for the study of muscular developmental genes.The expression of miR-2425 in the differentiation of bovine skeletal muscle satellite cells(MDSCs)was significantly decreased by preconcentration of miRNAs in the process of differentiation of bovine skeletal muscle.Suggesting that it plays an important role in MDSCs cell development and muscle formation.And through bioinformatics analysis,suggesting that it may promote the proliferation of bovine skeletal muscle cells,inhibit its differentiation.In particular,miR-2425 is a miRNA that is specifically expressed in cattle.In other mammals is not clear.There is no report on the biological function of miR-2425.Therefore,this study to miR-2425 as the object of study.The research is as follows:(1)The expression of miR-2425 in skeletal muscle satellite cells was detected by RT-PCR in different time.The expression of miR-2425 was detected by RT-PCR.The results showed that the expression of miR-2425 was the highest at 48 h of proliferation,and the expression level of miR-2425 gradually decreased with the prolongation of differentiation time.(2)miR-2425 mimics,mock control and miR-2425 inhibitors,inhibitor controls were transfected into MDSCs.EDU and flow results showed that compared with the control group, miR-2425 mimicry had a role in promoting proliferation of bovine skeletal muscle satellite cells.In contrast,after inhibition,the number of cell proliferation decreased.(3)The expression of CCNB1 and PCNA was detected by western blot.The results showed that the expression of CCNB1 and PCNA was up-regulated and decreased after the miR-2425 mimetic was transfected.(4)The expression of myosin heavy chain 3(MYH3)by immunofluorescence staining of desmin and western blot showed that miR-2425 mimics inhibited MDSC differentiation.While miR-2425 inhibitors promote MDSC differentiation.(5)The target genes of miR-2425 regulation were MYOG and RAD9 A by mir-base /Targetscan prediction.The luciferase reporter,RT-PCR and WB assay showed that miR-2425 directly targets the 3'-UTR region of RAD9 homologs A(RAD9A)and myoblastogenesis(MYOG)and negatively regulates their expression.(6)In order to further determine that RAD9 A inhibited MDSC proliferation,a RAD9 A overexpression vector was constructed and named pc DNA3.1-RAD9 A.The results of supplemental EDU showed that the number of proliferating cells in the overexpressed RAD9 A group was significantly lower than that in the control group compared with the control vector pcDNA3.1 empty vector.RAD9 A can be shown to inhibit the proliferation of MDSC.(7)miR-2425 is an intron miRNA and is located in the first intron of the bovine NCKAP5 L gene.The results of crisp Ri interferometry show that the expression of miR-2425 after transcription of host gene NCKAP5 L is also decreased.The dual luciferase reporter assay showed that miR-2425 was not a single promoter,and the combination of Crisp Ri interference experiments confirmed that miR-2425 was derived from introns that were transcribed by the host gene and were not transcribed as a single gene.In general,these data indicate that miR-2425 is a novel modulator of proliferation and differentiation of bovine MDSC and provides a better understanding of the biological function of miRNAs in this species.