The Molecular Mechanism Of MiR-24-3p/miR-200c-5p Regulating Myogenesis And Muscle Fiber Type Transformation

China has a long history of pig raising,and Chinese residents rely heavily on and consume pork.With the improvement of economic conditions and living standards,people’s requirements for the quality of pork are also increasing.The ability of myogenesis and the composition of muscle fiber types in pigs are key factors affecting the production and quality of pork.Myogenesis and muscle fiber type transformation are regulated by complex and delicate genetic mechanisms.As a non coding RNA,miRNA plays a crucial role in the growth and development of skeletal muscle.In the previous stage,our research group screened the key miR-24-3p from the whole genome and transcriptome sequencing data of the Tongcheng pig and Landwhite pig skeletal muscle.In addition,in order to study the effects of miRNA on skeletal muscle more deeply and extensively,this study further screened the important miR-200c-5p from C2C12 myoblast differentiation microarray data.However,the effects and underlying mechanisms of miR-24-3p and miR-200c-5p on myogenesis and muscle fiber type transformation remain unclear and need to be further studied.Therefore,this study aims to explore the functions and mechanisms of miR-24-3p and miR-200c-5p in regulating myogenesis and muscle fiber type transformation,To analyze the biological process of muscle growth and development from the perspective of miRNA.To provide new ideas for the improvement of meat quality of livestock and poultry,and the treatment of muscle-related diseases.The main results of this study are presented below:miR-24-3p regulates myogenesis and muscle fiber type transformation.It was found that the expression of ssc-miR-24-3p was higher in porcine longissimus dorsi muscle(fast muscle)than soleus muscle(slow muscle),and was upregulated during the differentiation of porcine skeletal muscle primary cells.Furthermore,q PCR,Western blot,CCK-8,and immunofluorescence assays showed that miR-24-3p overexpression inhibited the expression of proliferative marker genes KI67,PCNA,CDK4,Cyclin D1,and Cyclin E1,and proliferation rate in primary porcine skeletal muscle cells and C2C12 myoblasts.It up-regulated differentiation marker genes Myo D,My HC,Myo G,and promoted myotube fusion.It promoted the expression of the fast muscle fiber marker gene Myh4 and downregulated the slow muscle fiber marker gene Myh7.The effects of knocking down miR-24-3p on the proliferation,differentiation,and muscle fiber transformation of skeletal muscle cells were contrary to overexpression.In addition,the expression of mmu-miR-24-3p decreased first and then increased during skeletal muscle regeneration in mice.It indicated that mmu-miR-24-3p plays an crucial role in skeletal muscle regeneration and myogenesis.Subsequently,miR-24-3p overexpression downregulated the expression of Pax7,promoted the expression of Myo G and My HC,and the generation of muscle fibers in mouse skeletal muscle reconstruction.Moreover,miR-24-3p overexpression promoted the expression of Myh4 and increased the proportion of fast muscle fibers in mouse gastrocnemius muscle.miR-24-3p is conserved in pigs and mice.Through website prediction,MAPK14,NEK4,PSKH1,PIM1,and NLK were the target genes of mmu-miR-24-3p,and their expression were opposite to that of mmu-miR-24-3p in the process of mouse skeletal muscle regeneration.In addition,the expression levels of predicted target genes were higher in slow muscle fibers of pigs and mice than in fast muscle,contrary to miR-24-3p.Further dual luciferase assays proved the targeting relationship between miR-24-3p and MAPK14,NEK4,PSKH1,PIM1,and NLK.These results indicated that miR-24-3p is involved in the regulation of skeletal muscle generation and the transformation of fast and slow muscle fibers,which is conserved in function and regulatory mechanism among different species.miR-200c-5p regulates myogenesis and muscle fiber type transformation.The results showed that mmu-miR-200c-5p was down-regulated during C2C12 myoblast differentiation.The results of q PCR,Western blot,CCK-8,and immunofluorescence assays showed that miR-200c-5p overexpression inhibited the expression of differentiation marker genes Myo G and My HC,and myotube fusion;Promoting the expression of slow muscle fiber marker genes Myh7 and Myh2;It also promoted the expression of migration marker genes Plac8,Eno1,P2y6,Fak,Rac1,and migration behavior of C2C12 myoblasts.But the proliferation of C2C12 myoblasts was not affected.Knockdown of miR-200c-5p had opposite effects on differentiation,migration,and muscle fiber type transformation compared to overexpression.Further,Adamts5 was confirmed as a direct target gene of miR-200c-5p by website prediction,RIP,and dual luciferase assay.Adamts5 is highly expressed in mouse skeletal muscle,and its regulatory effects on C2C12 myoblasts migration and differentiation were opposite to miR-200c-5p.In addition,overexpression of miR-200c-5p rescued the regulation of Adamts5 on migration and differentiation of C2C12 myoblasts.These results indicated that miR-200c-5p is involved in skeletal muscle regeneration and fast-slow muscle fiber conversion by targeting Adamts5 expression and regulating skeletal muscle cell migration,differentiation,and muscle fiber type marker gene expression.In summary,two key miRNAs were screened from the previous sequencing data in this study.On the one hand,the regulatory effects of miR-24-3p and miR-200c-5p on skeletal muscle myogenesis and muscle fiber type transformation were verified.On the other hand,the target genes of miR-24-3p and miR-200c-5p were identified respectively.The regulatory mechanisms of these two miRNAs in skeletal muscle were analyzed.