Expression Of Bovine TDGF1 Gene Molecular Mechanism Effects In Myoblast Proliferation

Gene function and regulation mechanism that control beef muscle development are of the economic importance in livestock production to improve beef meat. Teratoma derivation growth factor(teratocarcinoma- derived growth factor 1, TDGF1) is a regulation of cell proliferation,survival, differentiation and migration of small molecule such as glycosyl phosphatidyl inositol protein(GPI). The TDGF1 as a cofactor in TGF-βsignaling pathways participated in the control of cell proliferation and apoptosis activation of multiple signaling pathways. Previously,scientific manuscript based on TDGF1 focused on tumor or embryonic development. However,recent study found that the TDGF1 is involved in regulation of skeletal muscle development. In mice, the TDGF1 was found to inhibit the MSTN signals and promote the development of skeletal muscle. Conversely, the cattle TDGF1 gene effects on muscle development are not clearly evaluated. In the current study, the bovine TDGF1 gene of myoblast proliferation and the TDGF1 eukaryotic expression vector was constructed. The expression of TDGF1 was evaluated in C2C12 cells to study their effects on cell proliferation and the regulation of MSTN function through the MSTN signaling pathways regulating myoblast proliferation. The aim of the current study was to evaluate the bovine TDGF1 gene and provide reference on its biological effect and mechanism in skeletal muscle.In order to find out the bovine TDGF1 affect myoblast proliferation. Using the directional cloning and cell transfection technique to obtained stable expression of bovine TDGF1 gene in C2C12 cell line,Western Blotting(WB) and fluorescence quantitative PCR technology detected TDGF1 in the expression of C2C12 cell line,determined by Methylthiazolyldiphenyl-tetrazolium bromide(MTT) method to detect C2C12 cells proliferation.The result indicated that build pIRES2-EGFP-TDGF1 eukaryotic expression vector successfully,bovine TDGF1 gene expression significant in C2C12 cells, successfully expressed TDGF1 protein and inhibited proliferation of C2C12 cells.In summary,built bovine TDGF1 gene expression vector successfully and TDGF1 gene can inhibit the myoblast proliferation and inhibition is the most significant when 30 h.In order to evaluate the TDGF1 interaction with the MSTN influence on the proliferation of C2C12 cells. Two related genes were used, the Myo D1 gene to evaluate C2C12 cells proliferation and Myf5 gene to evaluate if it can stabilize a cow TDGF1 of C2C12 cell line while an empty plasmid transfection was used as a control group and adding differentconcentration of MSTN protein in cells. Adding different concentration of MSTN protein, for both C2C12 cell proliferation inhibition,10μM MSTN is the strongest inhibitory effect.Transfection group pIRES2-EGFP-TDGF of C2C12 inhibitory effect is significantly higher than the empty plasmid group pIRES2-EGFP.The fluorescence quantitative PCR technique was used to detect MyoD1 with relative to expression of Myf5 gene. To study the C2C12 cells proliferation TDGF1 and MSTN gene transcription level, different MSTN protein concentration was detected by the MTT method. TDGF1 interact with MSTN’s influence on the proliferation of C2C12 cells. Western Blotting was used to measure different protein concentration, adding TDGF1 and Smad2 protein expression in C2C12 cells in mice. This study results indicate that the MSTN by TDGF1 activate its signaling pathways, start the Smad protein phosphorylation which lead to MyoD1 gene expression significantly lower and inhibits the proliferation of C2C12 cells.