Effect And Molecular Mechanism Of MiR-493-3p On Proliferation And Differentiation Of Porcine Skeletal Muscle Satellite Cells And Transformation Of Muscle Fiber Types

As the richest and most complex tissue in the human body,skeletal muscle is essential for the body’s health and normal function.In animal husbandry,the development of skeletal muscle is directly related to the production of meat,which is one of the most important economic characteristics.miRNAs(micro RNAs)play an important role in skeletal muscle cell growth,differentiation,myofiber type switching,and disease control,but many miRNA functions are still unknown.Previous studies have found that miR-493-3p is differentially expressed at different developmental stages in pig skeletal muscle and plays an important role,but the specific role is not yet clear.Our research group’s previous studies have shown that miR-493-3p is highly expressed in pig skeletal muscle tissue.Combined with previous studies indicating that miR-493-3p plays a crucial role in skeletal muscle development,it is necessary to deeply understand the mechanism of miR-493-3p’s action in skeletal muscle.In this experiment,the effects of miR-493-3p on the proliferation and differentiation of porcine skeletal muscle satellite cells and the transformation of muscle fiber types were deeply discussed.The target gene of miR-493-3p was screened by transcriptome sequencing and verified by diluciferase reporter experiment.To explore the mechanism of miR-493-3p on the myogenic process.To verify the role of miR-493-3p in the transformation of skeletal muscle fiber types at the living level.The following key findings were made:1.Expression pattern analysis of miR-493-3pmiR-493-3p is widely expressed in various tissues of pigs and is highly expressed in pig skeletal muscle.The expression level of miR-493-3p in Sol(mainly slow twitch)was significantly higher than that in LD(mainly fast twitch).In the proliferation stage of porcine skeletal muscle satellite cells,the expression of miR-493-3p decreased first and then increased.In the differentiation stage of porcine skeletal muscle satellite cells,the expression of miR-493-3p first increased and then decreased.2.miR-493-3p inhibits the proliferation of porcine skeletal muscle satellite cellsAfter overexpression of miR-493-3p,the number of S-phase cells in pig skeletal muscle satellite cells decreased significantly,and the proportion of Ed U-positive cells(P<0.05)decreased significantly.The m RNA levels and protein levels of the proliferative genes PCNA and Cyclin D were significantly reduced,and the expression of the proliferative-related gene p27 was significantly increased(P<0.05).After interfering with the expression of miR-493-3p,it promoted the proliferation of porcine skeletal muscle satellite cells.3.miR-493-3p promote the differentiation of porcine skeletal muscle satellite cellsAfter overexpression of miR-493-3p,the myotube generation capacity was significantly reduced,and the differentiation index decreased(P<0.05).The levels of m RNA and protein in My HC,Myo D and Myo G,myogenesis-related genes,were significantly increased,promoting myosat cell differentiation(P<0.05).Interfering with miR-493-3p gives the opposite result.Overexpression of miR-493-3p promoted the conversion of fast-twitch fibers to slow-twitch fibers in porcine skeletal muscle satellite cells,and significantly promoted the formation of slow-twitch fibers(P<0.05).After interfering with miR-493-3p,it inhibits the formation of slow-twitch fibers.5.miR-493-3p regulates the JNK pathway by targeting MKK7 to regulate skeletal muscle satellite cell proliferation and differentiationTranscriptome sequencing combined with diluciferase experiments determined that MKK7 was a candidate target gene for miR-493-3p;After overexpression or interference with miR-493-3p,MKK7 had a reverse trend.After MKK7 overexpression,the effect of overexpression miR-493-3p on the proliferation and differentiation of porcine skeletal muscle satellite cells was restored.Overexpression of miR-493-3p inhibits phosphorylation of JNK/MAPK,while interference with miR-493-3p reduces phosphorylation of JNK/MAPK.Overexpression of MKK7 significantly increased phosphorylation levels(P<0.05)of JNK/MAPK.In summary,miR-493-3p was involved in regulating the proliferation and differentiation process of porcine skeletal muscle satellite cells by targeting MKK7 and inhibiting the JNK pathway,and promoted the formation of slow twitch fibers in mouse skeletal muscle.These results enrich the development network of miRNA regulating skeletal muscle and provide a theoretical basis for improving pork quality.