The Role Of LncRNA-AK394747 Gene In The Proliferation And Differentiation Of Porcine Skeletal Muscle Satellite Cells

Skeletal muscle satellite cells are located between the surface of the myofibril membrane and the basement membrane and play an important role in the normal development and regeneration of muscles.Long non-coding RNAs(lncRNAs)are RNA molecules with a transcript length of more than 200 nt and low protein-coding potential.LncRNAs could regulate gene expression,participate in epigenetic modifications and affect the progression of various life activities,such as cell proliferation,differentiation,and apoptosis.In this study,we explored the function and the possible regulatory mechanism of a novel lncRNA-AK394747 in the process of proliferation and differentiation of in vitro isolated pig skeletal muscle satellite cells.The results are as follows:1.We successfully isolated skeletal muscle satellite cells by the method of two enzymes digestion,with high activity and proliferative capacity.After being differentially adherent purified,the cells were identified by using Pax7 gene.The results of immunofluorescence showed that the positive rate of cells was more than 95%.At the same time,the differentiation-inducing culture of isolated cells formed a large number of myotubes,which stably expressed the myogenic markers MyoD,MyoG and MyHc.2.The results of RACE showed that AK394747 was 1586 bp in full length without ability of proteins encoding,mainly present in the cytoplasm.It was specifically highly expressed in muscle tissues such as leg muscles and dorsal muscles,and at the same time the expression level increased with the differentiation of porcine skeletal muscle cells.3.The method of interference and overexpression were used to detect the role of AK394747 in the process of proliferation and differentiation in pig skeletal muscle satellite cells.The results of EdU,RTCA,and cell cycle tests showed that there was no effect of the AK394747 gene expression on the proliferation of skeletal muscle cells,and the gene expression of ki67,N-Ras and CDK family were not significantly changed.After knocking down the expression of AK394747,the mRNA and protein level of MyoD,MyoG and MyHc was significantly decreased,which were detected by techniques of QPCR,westren blotting,and immunofluorescence,indicating that AK394747 promoted the differentiation of porcine skeletal muscle cells.4.Four miRNAs which potentially bind to AK394747,such as ssc-miR-32,sscmiR-218,ssc-miR-218-3p and ssc-miR-301 were screened according to thebioinformatics prediction.After koncking down AK394747,the expression of pri-miR-32 and miR-32 was reduced.AK394747 was predicted to bind to Notch1 protein,as well as the result of Q-PCR shows that interference with AK394747 could inhibit the expression of NICD(Notch1 intracellular domain).In this study,The basic characterisc of porcine lncRNA-AK394747 and its function in the promotion of the skeletal muscle cells differentiation were illustrated.In addition,the possible role of porcine AK394747 was preliminarily investigated.The results lay a foundation for studying the mechanism of AK394747 in the development of skeletal muscle cells,and provideds a new idea for studying the regulatory mechanism of pig skeletal muscle development.