The Effect And Mechanism Of Long Noncoding RNA Lnc-mg To Promote Myogenesis

Purpose Study on the effect and explore the mechanism of long noncoding RNA lnc-mg act as a ce RNA to promote myogenesis.Method 1.lnc-mg knockdown vector and lnc-mg expression vector were constructed and transfected into myoblasts by lipofectamine3000.We used immunofluorescence method to investigate the number of My HC positive myotubes and q PCR to examine the expression of myogenic marker genes Myod and Myog.2.We used microarray analysis to screen the mi RNA regulated by lnc-mg by.3.We investigated whether lnc-mg had potential binding sites with mi R-125 b by dual-luciferase assay.4.We detected whether mi R-125 b bind to endogenous lnc-mg by biotin-labeled mi RNA capture and Ago2-IP.5.We detected the expression of Igf2 by Western Blot and ELISA.6.It is identified that lnc-mg function as a competing endogenous RNA for mi R-125 b by Ago2-IP,dual-luciferase assay and biotin-labeled mi RNA capture.Results 1.It suggests that knockdown of lnc-mg reduces the number of My HC positive myotubes and downregulates expression of myogenic marker genes Myod and Myog compaired to the control group.Moreover,overexpression of lnc-mg increases the number of My HC positive myotubes and upregulates Myod and Myog expression.2.Microarray analysis shows that mi R-125 b is down regulated when lnc-mg is overexpressed,while upregulated when lnc-mg is knocked down.The expression of lnc-mg is increased,while mi R-125 b is decreased in the process of myogenic differentiation.3.It shows that lnc-mg has potential binding sites with mi R-125b;Dual-luciferase assay suggests that the luciferase activity of Luc-lnc-mg is significantly reduced by mi R-125 b,while not affected by lnc-mg binding site mutated mi R-125 b.4.Ago2 immunoprecipitation confirm that lnc-mg and mi R-125 b are contained in the Ago2 complex;Biotin-labeled mi RNA capture assay reveal that lnc-mg can be captured by wild type mi R-125 b,while not lnc-mg binding site mutated mi R-125 b.5.Western Blot and ELISA show that Igf2,the target gene of mi R-125 b,is downregulated by lnc-mg knockdown and upregulated by lnc-mg overexpression.6.Ago2 immunoprecipitation confirm that overexpression of lnc-mg leads to the increased enrichment of Ago2 on lnc-mg,while substantially decreased enrichment on Igf2;Luciferase reporter assay shows that the luciferase activity of Igf2 3'UTR reporters is increased when wild-type lnc-mg overexpression but not upon mi R-125 b binding site mutated lnc-mg;Streptavidin capture analysis further suggests that the binding enrichment of mi R-125 b on Igf2 decreases with overexpression of wild-type lnc-mg,but not with mi R-125 b binding site mutated lnc-mg.Conclusion 1.It suggests that lnc-mg promotes myogenic differentiation of myoblasts.2.lnc-mg is involved in myogenic differentiation by regulating the expression of mi R-125 b.The expression of mi R-125 b was negatively related with lnc-mg expression.3.lnc-mg has potential binding sites with mi R-125 b.4.mi R-125 b can bind to endogenous lnc-mg.5.lnc-mg can modulate the expression of Igf2,which is the target gene of mi R-125 b.6.lnc-mg acts as a ce RNA in the process of promoting myogenesis.That it is,lnc-mg results in decreased mi R-125 b by acting as a molecular sponge,which subsequently controls the expression of Igf2.