Effect Of MiR-668-3p On Proliferation And Differentiation Of Myoblast Cells And Its Molecular Mechanism

Skeletal muscle is the most abundant and complex tissue in mammals,and its normal function is crucial to body health.This process involves the normal operation of various signaling pathways and genes.As one of the important organs for maintaining body operation,skeletal muscle is not only closely related to human physiological diseases,but also closely related to the meat yield and quality of livestock.Micro RNA(miRNA)is a group of short non-coding RNAs,consisting of about 20 nucleotides,which control gene expression through targeted m RNA after transcription and play a role in skeletal muscle cell proliferation,differentiation,muscle fiber type transformation and disease regulation.Since miRNA was first discovered 20 years ago,the miRNA population has been continuously expanding and many miRNAs still have unknown functions due to the temporal and tissue specificity of miRNA.There are not many existing research results on miR-668-3p,mainly focusing on ischemic reperfusion injury,and a few studies on cancer,but not in-depth.miR-668-3p is highly expressed in skeletal muscle and differentially expressed in fast and slow myofibers.However,its role in skeletal muscle fiber type transformation has not been reported yet.Therefore,it is necessary to further understand the role of miR-668-3p in skeletal muscle.In this study,mouse myoblast cell line(C2C12 cells)was used as the research model to explore the effect of miR-668-3p on myoblast process and the transformation of muscle fiber type.The target genes of miR-668-3p were predicted by online prediction software and verified by dual-luciferin reporting assay.To explore the potential mechanism of miR-668-3p on myogenesis.The main research results are as follows:1.Expression pattern of miR-668-3pmiR-668-3p was widely expressed in all tissues of mice,with the highest expression level in skeletal muscle,followed by adipose tissue.The expression level of miR-668-3p in EDL(mainly fast myofibers)was significantly higher than that in sol(mainly slow myofibers).At the proliferation stage of C2C12 cells,the expression level of miR-668-3p first decreased and then increased.At the differentiation stage of C2C12 cells,the expression pattern of miR-668-3p was firstly increased and then decreased.2.miR-668-3p inhibited the proliferation of C2C12 cellsOverexpression of miR-668-3p significantly inhibited the number of S-phase cells in C2C12 cells and reduced the proportion of Ed U positive cells.The m RNA and protein levels of proliferation-related genes Cyclin E and Cyclin D were significantly decreased,while the inhibitory genes p27 and p21 were significantly increased.This result was reversed after interference with miR-668-3p.3.miR-668-3p inhibited the differentiation of C2C12 cellsOverexpression of miR-668-3p significantly reduced the myoduct formation ability and differentiation index.The protein and m RNA levels of myogenesis related genes My HC,Myo D and Myo G decreased and inhibited cell differentiation.Interference with miR-668-3p yielded opposite results.Overexpression of miR-668-3p inhibited the formation and expression of fast and slow myotubes.Interference with miR-668-3p can promote the formation of slow myotubes,promote the expression of slow myofiber gene and inhibit the expression of fast myofiber gene.4.Appl1 is the target gene of miR-668-3pOnline prediction software results showed that Appl1 was a candidate target gene of miR-668-3p,which was verified by dual luciferase assay.After miR-668-3p overexpression or interference,the change trend of Appl1 was opposite.The inhibitory effect of overexpression of miR-668-3p on proliferation and differentiation of myoblasts was recovered after overexpression of Appl1.5.miR-668-3p inhibits myoblast proliferation and differentiation by targeting Appl1 and regulating MAPK pathwayOverexpression of miR-668-3p inhibited the phosphorylation of p38 MAPK,while interference of miR-668-3p reduced the phosphorylation of p38 MAPK.After Appl1 overexpression,the phosphorylation level of p38 MAPK was significantly increased.In summary,we found that miR-668-3p,as a negative regulator in myogenesis,inhibits myoblast proliferation and differentiation by targeting Appl1 and down-regulating MAPK pathway.In terms of myofiber type transformation,interference with miR-668-3p can promote slow myotubes.These findings contribute to the enrichment of skeletal muscle regulatory network at the miRNA level,and suggest that miR-668-3p/Appl1/MAPK pathway may serve as a potential target for studying skeletal muscle growth and development,providing new ideas for human medicine and animal husbandry.