The Effect Of SkMLCK Gene On Muscle Fiber Components And Signal Pathways In Myoblast Cells C2C12

Objective In order to analyze the expression of key molecules in the muscle fiber and the signal pathways by real-time quantitative PCR and Western blot technique,the mouse C2C12 myoblast cell lines of sk MLCK gene.This experiment provided a preliminary study of sk MLCK on muscle fiber composition and related signal pathways and lays the foundation for other related researches.Methods This research group recovered the sk MLCK stabled high expression transfected cell lines which had been obtained in early stage and expended culture.The highly efficient sh RNA was selected and transfected it into C2C12 cells,and the stably transfected cell lines were obtained by screening with puromycin.sk MLCK,CFL2,Muscle fiber components(MLC,?-actin,My HC slow,My HC 2a,My HC 2x and My HC 2b)and key factors in signal pathway(Ca M,p38,AMPK?2,Rho A and sk MLCK)m RNA and protein expression level were detected and statistical analyzed by the method of real-time quantitative PCR and Western blot technique?Results 1.The experiment of sk MLCK high expression result by the method of real-time quantitative PCR showed that m RNA levels of Myhc slow,Myhc 2a,MLC in the muscle fiber components comparing with the blank control group were improved significantly or very significantly(p<0.05 or p<0.01)and ?-actin,Myhc 2x,Myhc 2b were decreased significantly(p<0.01).And the key factors in signal pathway such as MEF2 C,Ca M,p38,AMPK?2,Rho A were down-regulated significantly or very significantly(p<0.05 or p<0.01).The expression of CFL2 were decreased significantly or very significantly in each group(p<0.05 or p<0.01).The experiment result of sk MLCK high expression by the method of Western blot technique indicated that protein expression of Myhc 2a and MLC in the muscle fiber components comparing with the blank control group were up-regulated significantly(p < 0.01)and ?-actin,Myhc slow,Myhc 2x,Myhc 2b were down-regulated significantly or very significantly(p<0.05 or p<0.01).The key factors in signal pathway such as MAPKp38 and Rho A were improved significantly(p<0.01).MEF2 C,Ca M and AMPK?2 were down-regulated significantly or very significantly(p < 0.05 or p < 0.01).The expression of CFL2 were decreased significantly or very significantly in each group(p<0.05 or p<0.01).2.The experiment of sk MLCK low expression result by the method of real-time quantitative PCR showed that m RNA levels of ?-actin,MLC,Myhc 2x,Myhc 2b in the muscle fiber components comparing with the blank control group were improved significantly or very significantly(p<0.05 or p<0.01)and Myhc slow and Myhc 2a were decreased very significantly(p<0.01).Meanwhile,the key factors in signal pathway such as AMPK?2 and Rho A were up-regulated very significantly(p<0.01).The expression of CFL2 were increased significantly or very significantly in each group(p<0.05 or p<0.01).The experiment result of sk MLCK low expression by the method of Western blot technique indicated that protein expression of ?-actin,MLC,Myhc 2x,Myhc 2b in the muscle fiber components comparing with the blank control group were improved significantly or very significantly(p<0.05 or p<0.01).Meanwhile,the key factors in signal pathway such as AMPK?2 and Rho A were up-regulated very significantly(p<0.01).The expression of CFL2 were increased significantly or very significantly in each group(p<0.05 or p<0.01).3.According to the results of the high and low expression of all protein expression of stably transfected cell lines.The correlation analysis results of Significant(p<0.05)or very significant(p<0.01)shows that sk MLCK was highly correlated with Rho A(p<0.05),and the correlation coefficient was 0.818,the relationship of Rho A and sk MLCK was not a simple liner,but a curvilinear.Conclusions 1.sk MLCK affects the composition of muscle fibers by changing the Rho A signaling pathway in C2C12.2.sk MLCK can inhibit the growth and development of muscle fiber.3.sk MLCK inhibited the expression of CFL2.And it also affects other signal pathways(AMPK,Ca M and MAPK).