Effectof MiR-181a On The Differentiation Of Bovine Skeletal Muscle Satellite Cells

Micro RNA is one of the important regulators of gene expression,which belongs to the non-coding small RNA molecule,which has the function of regulating cell proliferation,differentiation and apoptosis.In the past,people have studied the expression of muscle genes at the transcriptional level.Recently,miRNAs have been found to act as transcriptional regulators,and have some regulation in skeletal muscle development,so that people can have a deeper understanding of muscle development.In view of the important role of miRNA in the differentiation of skeletal muscle satellite cells,this study investigates the expression and regulation mechanism of miRNAs in the process of differentiation of bovine skeletal muscle satellite cells.The miRNA-181 a micro RNA was found to be highly expressed in the process of differentiation of bovine skeletal muscle satellite cells when sequencing of different time different miRNAs during the differentiation of bovine skeletal muscle satellite cells by high-throughput deep sequencing(Hi Seq deep sequencing).We speculate that the micro RNA may be involved in the regulation of bovine skeletal muscle satellite cells.We used real-time quantitative RT-PCR(q RT-PCR)to determine the expression of miRNA-181 a at different time and found results consistent with the high-throughput sequencing.So this experiment to miR-181 a as a research object.The possible target genes of miRNA-181 a were analyzed by bioinformatics,and it was found that HOX-A11 might be one of the target genes.HOX-A11 is a gene that inhibits cell differentiation.By double luciferase reporter assay,immunofluorescence,overexpression and inhibition,q RT-PCR,Western blot and other techniques,the effect of miR-181 a on the expression of HOX-A11 and post-transcriptional level of differentiation-related genes was studied.It was found that miRNA-181 a could target HOX-A11 and regulate the differentiation of bovine skeletal muscle satellite cells.The effect on the differentiation of bovine skeletal muscle satellite cells is of great significance to improve the quality of beef and increase the economic value.The results are as follows:In the bovine skeletal muscle satellite cells,the expression of miR-181 a was detected by Real-time PCR,and the expression of miR-181 a increased significantly with time.The expression of HOX-A11 protein was detected by Western blot.The expression of HOX-A11 protein was decreased with the increase of time.The expression level of miR-181 a was inversely proportionalto the expression level of HOX-A11 protein,which was the common miRNA-target gene interaction.The number of myocardium transfected with miR-181a-M mimics was significantly higher than that of the miR-181a-M-NC control group by measuring the number of myocardium in the cells by immunofluorescence.The miR-181a-I inhibitor was significantly lower than that of the miR-181a-I-NC control group,and we observed morphologically more intuitively that miR-181 a promoted the differentiation of bovine skeletal muscle satellite cells.In the dual luciferase reporter system,when the miRNA-181 a was able to bind to the 3'-UTR of the HOX-A11 gene,the luciferase activity was significantly decreased.When the miRNA-181 a and the mutated 3'-UTR were bound,the luciferase activity There is no decline,further indicating HOX-A11 is the target gene of miR-181 a.The expression of HOX-A11 protein in miR-181a-M mimics was significantly decreased in the bovine skeletal muscle satellite cells,and the expression of miR-181a-I inhibitor HOX-A11 protein was significantly increased,which indicated that miRNA-181 a can negatively regulate the expression of HOX-A11 gene.Mitral muscle satellite cells transfected with miR-181a-M mimics were significantly increased in the expression of muscle differentiation proteins such as Myo D,Myo G and MYH3,and the expression of miR-181a-I inhibitor was significantly decreased,it was confirmed that miR-181 a could promote cell differentiation.