MiR-92a-3p Inhibits C2C12 Migration By Targeting Itga6

Skeletal muscle satellite cells are myogenic stem cells which located between the muscle fiber membrane and the basement membrane.Skeletal muscle satellite cells have the potential for proliferation and differentiation.In spine animals,skeletal muscle satellite cells play an important role in the growth and damage repairing of skeletal muscle.C2C12 cell is a kind of mouse myoblast cell lines and have been widely used in the study of myogenic processes.miRNAs are short-chain non-coding RNAs that play important roles by regulating the expression of target genes.A large number of studies have shown that miR-92a-3p can regulate cell proliferation,migration and adhesion,however,the function of miR-92a-3p in myoblasts has not been reported.In order to clarify the function of miR-92a-3p in myoblasts,this study investigated the effects of miR-92a-3p on the proliferation,differentiation,migration,adhesion and spreading of C2C12 cells by overexpressing and interfering miR-92a-3p.The target gene of miR-92a-3p was predicted by bioinformatics method.The overexpression vector of target gene was constructed to verify the targeting relationship.And then,we overexpressed the target gene in C2C12 cell to investigate the function of targeted gene.At last,the recovery verification was performed in C2C12 cells.The main results are as follows:1.Effect of miR-92a-3p on proliferation of C2C12 cells.The proliferation of C2C12 cells was detected by Ed U method and flow cytometry at 24 h and 48 h after overexpressed miR-92a-3p.The results showed that miR-92a-3p did not affect C2C12 cell proliferation.2.Effect of miR-92a-3p on the differentiation of C2C12 cellsAfter overexpression of miR-92a-3p,C2C12 cells were grown to confluence and induced differentiation.Myosin immunofluorescence was performed at 3,5,7 and 9 days after induction.q RT-PCR was used to identify the mRNA levels of Myogenin and Myosin.The results showed that miR-92a-3p did not affect C2C12 cell differentiation.3.Effects of miR-92a-3p on migration,adhesion and spreading of C2C12 cellsC2C12 cell migration,adhesion assay and spreading assay were used to detect C2C12 cell adhesion and spreading 24 hours after miR-92a-3p overexpression and interference,respectively.The results showed that miR-92a-3p inhibited migration,adhesion and spreading of C2C12 cells.4.miR-92a-3p target gene prediction and verificationBioinformatics methods were used to predict target genes of miR-92a-3p.The targeting relationship was verified by dual luciferase reporter system,q RT-PCR and Western blot.The results show that miR-92a-3p is able to directly target Itga6 gene.5.Effect of Itga6 on migration,adhesion and spreading of C2C12 cellsThe overexpression vector pcDNA3.1-Itga6 was successfully constructed.The Itga6 gene was successfully overexpressed in C2C12 cells.The scratch test,live cell workstation,adhesion assay and spreading assay showed that Itga6 promoted the migration,adhesion and spreading of C2C12 cells.6.Functional recovery testOverexpression of miR-92a-3p inhibits migration,adhesion and spreading of C2C12 cells,while overexpression of miR-92a-3p and Itga6 can reverse the inhibition effect.While,the reversal degree of miR-92a-3p and Itga6 co-overexpression does not exceed the reversal degree of overexpressing Itga6 alone.miR-92a-3p inhibits the migration,adhesion and spreading of C2C12 cell by targeting Itga6.