The Influence And Primary Molecular Mechanism Of CFL2 Over Expression On Muscle Fibers In Myoblast

ObjectiveIn order to analyze the influence of CFL2 over expression on muscle fibers and related signal pathway, the mouse myoblast C2C12 strains of CFL2 over expression were established. This experiment would provide a primary molecular mechanism for the function of CFL2 on skeletal muscle and other research.MethodThe coding sequence of porcine CFL2 was inserted into p MD18-T vector and the CFL2 fragment was amplified and inserted into the eukaryotic expression vector p EGFP-N1. Then the recombinant was transfected into mouse myoblast C2C12 by liposomes and the stably transfected cell lines were built by G418 screening for 14 d. The expression of m RNA and protein for CFL2, skeletal muscle components(My HC, MLC and α-actin), and key factors in signal pathway(sk MLCK, MEF2 C, AMPKα2, MAPK p38, Ca M and Rho A) were detected and statistical analyzed by the method of real time RT-PCR and Western blot.Results1. The p EGFP-N1-CFL2 recombinant was constructed successfully and 3 stably transfected cell lines were established with different expression of CFL2.2. The experiment result by the method of real time RT-PCR showed that m RNA expression of My HC and α-actin in cell lines with over expression of CFL2 were increased and MLC decreased significantly. Meanwhile, the key factors in signal pathway, sk MLCK, MEF2 C, MAPK p38, Ca M and Rho A all showed up-regulated and only AMPKα2 down-regulated significantly.3. The results in protein expression by Western blot revealed that almost all proteins(My HC2 a, My HC2 b, MLC, α-actin, sk MLCK, MEF2 C, AMPKα2, MAPK p38, Ca M and Rho A) were increased significantly and only My HC2 x and My HC slow decreased significantly.4. The mathematical models for the protein levels between CFL2 and My HC slow, My HC 2x, α-actin and Ca M were established.ConclusionsThe over-expression of CFL2 in C2C12 increased the componants of muscle fibers(My HC, MLC and α-actin) and improved its growth. Meanwhile, it also changed the type of muscle fibers. The mechanism may involve 4 signal pathways. There were mathematical models for the protein levels between CFL2 and My HC slow, My HC 2x, α-actin and Ca M.