| Micro RNAs(miRNAs),as a class of endogenous single stranded non coding RNAs,have short chain length(22-25 nt)and high conservation among species.They can regulate the generation of skeletal muscle and maintain the structure and function of tissues and organs.After analyzing the data,we used q RT-PCR to obtain the expression profile of miR-424-5p in Xuhuai goat at different growth stages(adult and fetal stages)and 8 tissues.Finally,miR-424-5p was taken as the research object to explore how it regulates the proliferation and differentiation of skeletal muscle cells.In addition,the potential target gene of miR-424-5p of Hsp90aa1 gene was screened and verified based on the prediction of targetscan database and transcriptome analysis.Finally,the effect of Hsp90aa1 on the proliferation and differentiation of myoblasts was further studied.The results are as follows:1.Expression profile of miR-424-5p in different tissues of Xuhuai goatThe results of q RT-PCR showed that miR-424-5p was expressed in heart,liver,spleen,lung,kidney,small intestine,breast muscle and leg muscle of Xuhuai goat,and the expression level of miR-424-5p in breast muscle and leg muscle of fetal sheep was significantly lower than that of adult sheep.Therefore,it is speculated that miR-424-5p may be involved in regulating the growth and development of Xuhuai goat skeletal muscle development process.2.miR-424-5p can promote the proliferation of C2C12 cellsIn this study,the constructed miR-424-5p overexpression vector and the synthesized NC,miR-424-5p mimics,anti NC and miR-424-5p inhibitors were transfected into C2C12 cells respectively.By q RT-PCR and WB,the data showed that:after overexpression of miR-424-5p,the expression levels of proliferation marker genes(CDK1 and PCNA)were significantly up-regulated;when the expression of miR-424-5p was inhibited,the expression levels of CDK1 and PCNA were down regulated.In addition,the results of CCK-8 cell activity test and edu cell proliferation test showed that miR-424-5p promoted the proliferation of C2C12 cells.3.miR-424-5p can inhibit the differentiation of C2C12 cellsIn this study,the constructed miR-424-5p overexpression vector and the synthesized NC,miR-424-5p mimics,anti NC and miR-424-5p inhibitors were transfected into C2C12 cells,cultured and induced to differentiate.The detection methods included q RT-PCR and WB analysis.The data showed that when miR-424-5p was overexpressed,the relative expression of differentiation marker genes(Myo G and Myo D)decreased significantly;on the contrary,the relative expression levels of differentiation marker genes were significantly increased.At the same time,the state of myotubes in the process of cell differentiation was observed by cell immunofluorescence assay,which verified the inhibitory effect of miR-424-5p on myoblast differentiation.4.Hsp90aa1 is the target gene of miR-424-5pFive potential target genes(FNTA、FGF18 、MBNL2、NEBL、Hsp90aa1)of miR-424-5p were screened by transcriptome analysis combined with targetscan and David database.After successfully constructing wild and mutant dual luciferase vectors,they were cotransfected into HEK293 T cells with NC and miR-424-5p mimics,respectively After transfection,Hsp90aa1 was identified as a potential target gene of miR-424-5p.5.Interference with Hsp90aa1 can promote the proliferation of C2C12 cells and inhibit the differentiation of C2C12 cellsIn order to study the effect of Hsp90aa1 on the proliferation and differentiation of C2C12 cells,the expression of Hsp90aa1 was interfered,and then si-NC and si-Hsp90aa1 were transfected into C2C12 cells respectively to induce cell proliferation or differentiation The relative expression levels of proliferation marker genes(CDK1and PCNA)and differentiation marker genes(Myo G and Myo D)at m RNA and protein levels were detected by Blot assay.Combined with CCK-8 and Ed U assay,the final results showed that the interference of Hsp90aa1 expression could promote the proliferation of C2C12 cells and inhibit their differentiation.In conclusion,miR-424-5p can regulate the proliferation and differentiation of skeletal muscle cells by targeting Hsp90aa1. |
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