| ObjectiveIn order to analyze the expression of key molecules in the muscle fiber and four signal pathways by real-time quantitative PCR and Western blot technique,the mouse myoblast C2C12 cell lines of MEF2C gene low expression were established.This experiment provides a basis for the further study on the influence of MEF2C gene low expression on muscle fiber composition and related signaling pathway.MethodsThree RNA interference sequence targeting porcine MEF2C gene were designed and synthetized by using RNAi technology and construction of RNA interference vectors(shRNA).Then they were instantaneously transfected into mouse C2C12 cells.The expression status of mRNA of MEF2C in cells was determined by real-time quantitative PCR and the expression status of protein of MEF2C in cells was determined by Western blot technique;Moreover,the highly efficient shRNA was selected and transfected it into C2C12 cells,and three mixed cell clones with low expression of MEF2C were obtained;MEF2C,CFL2,Skeletal muscle components(MLC,?-actin,MyHC slow,MyHC 2a,MyHC 2x and MyHC 2b)and key factors in signal pathway(Ca M,p38,AMPK?2,RhoA and skMLCK)mRNA and protein expression level were detected and statistical analyzed by the method of real-time quantitative PCR and Western blot technique.Results1.Three RNA interference vectors(shRNA)targeting of porcine MEF2C gene had been constructed successfully.The shRNA vectors were transfected instantaneously into C2C12 cells.Respectively,the inhibition rates on mRNA of MEF2C were 67.09%±5.68%,59.70%±5.21% and 68.80%±5.78% and the inhibition rates on protein of MEF2C were 19.33%±1.62%,17.58%±2.80% and43.81%±0.20%.Finally,the interference of shRNA-3 in the expression of MEF2C is best.2.The real time PCR results showed that mRNA levels of ?-actin,MyHC slow,MyHC 2x,MyHC 2b in the muscle fiber components comparing with the control group were improved significantly or very significantly(p<0.05 or p<0.01)and MLC,MyHC 2a were decreased significantly(p<0.01).Meanwhile,the expression of key factors in signal pathway such as Ca M,p38,AMPK?2 and Rho A were down-regulated significantly or very significantly(p < 0.05 or p <0.01).The expression of skMLCK was not significant in each group(p>0.05);The expression of CFL2 were decreased significantly or very significantly in each group(p<0.05 or p<0.01).3.The Western blot results indicated that protein expression of MLC,MyHC slow and MyHC 2a in the muscle fiber components comparing with the control group were down-regulated significantly(p < 0.01)and ?-actin,MyHC 2x and MyHC 2b were up-regulated significantly or very significantly(p<0.05 or p<0.01);The key factors in signal pathway such as p38 were improved significantly.The other four components(Ca M,AMPK?2,RhoA and skMLCK)were not significant(p>0.05);CFL2 were up-regulated significantly or very significantly(p<0.05 or p<0.01),4.The related mathematical models between MEF2C and p38,CFL2 and MyHC 2b were established respectively.Statistical analysis manifested that the mRNA expression of the genes was not consistent with protein in this study,which demonstrated that the process of translation from mRNA to protein was the key process of gene expression in C2C12 cell line.5.The results showed the expression of MEF2C mRNA was positively correlated with its protein significantly(P<0.01),and r2=0.985;MEF2C mRNA was extremely significant negative correlation with CFL2 protein(P<0.01),and r2=0.962;AMPK?2,CaM and p38 mRNA expression and MEF2C protein expression were significant or extremely significant positive correlation(P<0.05 or P<0.01).There were significant or extremely significant correlation between the rest of the gene mRNA and protein.ConclusionsThe low expression of MEF2C in C2C12 cells decreased the expression of MLC and MyHC 2a,while the expression of ?-actin,MyHC 2b and MyHC 2x were increased,which illustrated that the expression of MEF2C affected the composition of muscle fiber.The mechanism may involve four signal pathways,such as AMPK,CaM,MAPK and Rho. |
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