Primary Research Of Mechanism Of CFL2 Down-regulation On Muscle Fiber In Mouse Myoblast C2C12

Objectives Short hairpin RNA recombinant vectors of CFL2 gene were designed and transfected into muscle C2C12 cells. Stably cell lines were screened and used to do research on the effect and mechanism of CFL2 low expression on components of muscle fibers and key factors in signal pathway. This study would provide a new sight for further research of CFL2 function.Methods Then C2C12 cells were transfected by sh RNA recombinant vectors targeting CFL2 gene according to RNAi method. The m RNA and protein expression levels of CFL2, component of muscle(MLC、α-actin、My HC2b、My HC2 x 、 My HC2 a �'� My HC slow) and the key factors of signal pathway(MEF2C、sk MLCK、Rho A、AMPKα2、p38�'�Ca M) in the low expression groups were detected by Real-time PCR and Western blot.Results 1. Three recombinant plasmid vectors named CFL2-sh RNA for silencing CFL2 gene were constructed successfully. 2. Three CFL2-sh RNA vectors were transfected stably into mouse myoblast C2C12 cells respectively. The m RNA and protein level of CFL2 were down regulated differently by real-time PCR and Western blot. 3. In the low expression groups, m RNA levels of MLC as composition of fiber showed up-regulated significantly(p<0.05). The key factors in signal pathway such as MEF2 C, Ca M, P38 and AMPKα2 were decreased and sk MLCK increased significantly(p < 0.01). The fiber type of My HC slow, My HC2 a and My HC2 x also down-regulated significantly(p<0.01). 4. The analysis for protein expression by Western blot in the low expression group showed that in one of three transfected cell strains, MLC and α-actin were increased as well as My HC2 b, My HC2 a and My HC2 x decreased significantly(p< 0.01). The protein levels for key factors(MEF2C and Ca M) in three transfected cell strains were down-regulated significantly(p<0.05 or p<0.01). The other four factors(p38, Rho A, AMPKα2 and sk MLCK) were insignificant(p>0.05). 5. The statistic analysis showed that there were significantly positive or negative relationships betweem CFL2 and α-actin, MLC, MEF2 C, Ca M, sk MLCK, My HC2 b, My HC2 a or My HC2x(p<0.05 或 p<0.01).Conclusions 1. CFL2 gene silencing caused low expression of My HC2 b, My HC2 a and My HC2 x significantly. However, α-actin and MLC showed up-regulated significantly just in extremly low level of CFL2. 2. MEF2 C and Ca M low expression followed the down-regulation of CFL2 significantly. 3. There were significantly positive or negative relationships betweem CFL2 and α-actin, MLC, MEF2 C, Ca M, sk MLCK, My HC2 b, My HC2 a or My HC2 x.