Identification And Analysis Of MiRNAs Related To Wnt/?-catenin Signaling Pathway In Bovine Skeletal Muscle Satellite Cells Adipogenic Induction

Differentiated skeletal muscle satellite cells(SMSCs)are a population of multipotent cells and not only exist in the infancy of animal but adult mammalian body.Previous study indicated that Wnt/?-catenin signaling is involved in regulation of multipotent cells differentiation,and is essential for the regulation of adipogenesis.MicroRNAs(miRNA)are endogenous non-coding RNA,and play an important role in the formation of body fat.However,the differential expression of miRNAs of SMSC adipogenesis induction and role of these miRNAs in the wnt signaling has received little attention.1.Isolation and culture of bovine muscle satellite cells.Here we adopts hind leg skeletal muscle from beef cattle,and cutted it into patch which used mechanical separation,digested it into single cell by collagenase and typsin,purified by differential adherent method,thereby we got the skeletal muscle satellite cells.Based on myoblast induction shows,it appeared some muscle fibers in the second day of induction,but in the fourth day,there has large number of muscle fibers,and began to merge into thick mucle tube.When we used Oil Red O staining the adipogenesis induction cells,lipid droplets were visualized by Oil Red O staining,which shows it keep a good state of differetiation.By immunofluorescence verification specificity gene Pax7,results shows that more than 95%cells positive expression.These results confirmed that the skeletal muscle satellite cells can be used for the next experiment.2.MicroRNA expression profiles of Wnt/?-catenin signaling in SMSCs adipogenic induction.In this study,bovine skeletal muscle satellite cells(bSMSCs)was cultured and adipogenic induction.Wnt/?-catenin signaling pathway was activated by LiCl.Then,Profile of differentially expressed miRNA and the target gene analysis were investigated by microarray and bioinformatics.The data identified 29 differentially expressed miRNAs in 783 miRNAs which were changed significantly(fold change>2),12 were up-regulated and 17 were down-regulated.Using bioinformatics methods,24 of 29 miRNAs were involved in the fat metabolism,cells differentiation,development of the immune system,in 315 potential target genes,39 target genes were associate with adipogenesis and metabolism,immune response and stem cell differentiation.GO and KEGG pathway showed that these different expressed miRNAs were involved in different pathway and target genes which may differently regulate the SMSC differentiation into adipocyte.Taken together,our study provided the first evidence for miRNA differentially expressed in bSMSC adipogenic differentiation which influenced by Wnt/?-catenin signaling pathway,and also provided an overview of miRNAs expression profile and built an interaction between miRNAs and target genes,and that will lead to further understanding of these miRNAs which has important regulating function in Wnt/?-catenin signaling pathway in the process of SMSC adipogenic differentiation.