Influence Of MiR-206Gene On The Differentiation Of Bovine Skeletal Muscle Satellite Cells

MicroRNAs (miRNAs) are a class of evolutionally conserved noncoding RNAs that are about22nucleotides (nt) in eukaryotes, specific binding with the target mRNA3ˊuntranslated regionand negatively regulate gene expression by promoting degradation of target mRNAs or inhibitingtheir translation. MicroRNAs are an important regulator of post-transcriptional, which regulateexpression of gene. Many studies have revealed that miRNAs play crucial roles in a broad range ofbiological processes that including early development, cell proliferation, apoptosis and celldifferentiation. With more and more microRNAs have been found and explained their function andregulation pathway. MicroRNAs have become a hot research field of biology. Studies have shownthat microRNAs also played a key role in muscle development. The expression of miRNAs havetissue specificity in vivo, microRNAs of muscle-specific expression were named MyomiRs bysome scholars, which including miR-1, miR-133a, miR-206and so on. Most of the MyomiRs areexpressed in cardiac and skeletal muscle, but miR-206is unique,because it is only expressed inskeletal muscle. so muscle-specific miR-206as the research object in this experiment.In this paper, primary cultured of bovine skeletal muscle satellite cells as experimentalmaterial, we detect the expression level of miR-206in normal cultured bovine skeletal musclesatellite cells and differentiation in different periods and eukaryotic expression vector of miR-206by transfection into the bovine skeletal muscle satellite cells by Real Time PCR, ResearchmiR-206on the effect of in bovine skeletal muscle satellite cells differentiation, the resultsshowed:The combination of collagenase XI with trypsin digestion and after differential adhesion wereable to successfully isolated primary bovine skeletal muscle satellite cells. The cells growthcondition were good and had highly differentiation efficiency. When the cells were induced in2%horse serum, and the cells have began to differentiate and fused to multinucleated myotubes, andthe immunofluorescence staining of skeletal muscle molecules revealed the positive expression ofthe cells.The expression of miR-206was detected by real-time PCR at various differentiation stages ofbovine skeletal muscle satellite cells, the results showed that the expression levels of miR-206wasthe lowest in undifferentiated cells. With the prolongation of differentiation time and theexpression of miR-206increased gradually, it was the highest in the fifth day. By transfection of pcDNA3.1(+)-miR-206eukaryotic expression vector compared with the control group,over-expression of miR-206promoted differentiation of bovine skeletal muscle satellite cells.C2C12myoblasts were an ideal differentiation model in vitro. We detected the expression ofmiR-206during different stages of C2C12myoblasts differentiation, the experimental resultsshowed that the expression levels of miR-206was the highest in fifth days.We predicted that the target gene of miR-206was PAX3by Bioinformation. And through theRT-PCR amplified3’-UTR region of PAX3, double fluorescence report showed that PAX3was thetarget gene of miR-206.The results of the experiments explored the role of miR-206in bovine skeletal musclesatellite cells, For the future study the function and regulation mechanisms of MyomiRs in bovinemuscle development, which provide a theoretical basis and the experimental basis.