Study On The Effect Of IGF1 And MSTN On The Proliferation Of Myogenic Cells In Guanling Bovine

Insulin like growth factor 1（IGF1）and muscle growth inhibitor（Myostatin,MSTN）,as regulators of muscle growth in animal organisms,play an important role in the proliferation of myogenic cells.In this study,we investigated the effects of IGF1 and MSTN genes on the proliferation of myogenic cells in Guanling cattle by molecular cloning,CCK8,qPCR technique,cell scratching and flow cytometry,to further understand the muscle growth and development pattern of Guanning cattle and to provide basic data reference at the cellular level for the improvement of beef quality in Guanling cattle.1.Study on the effect of IGF on the proliferation of myogenic cells in Guanling cattle.The sequence of IGF1 gene was analyzed by bioinformatics such as a mino acid composition analysis and protein structure analysis to investigate the difference of m RNA expression of IGF1 gene in various tissues of Guanling cattle.skeletal muscle myogenic cells of Guanling cattle were cultured and identified for purity.pEGFP-C1-IGF1 overexpression vector and pGPU6-GFP-Neo interference vector were constructed.CCK8,qPCR,cell scratching and flow cytometry were used to detect the effect of IGF1 gene on proliferation of Guanling cattle myogenic cells.qPCR,cell scratching and flow cytometry were used to detect the effects of IGF1 gene on the proliferation of Guanling bovine myogenic cells.The results showed that IGF1 gene expression was the highest in liver,which was significantly higher than other tissues（P<0.01）.The molecular formula of IGF1 protein was C₇₄₄H₁₁₈₆N₂₁₄O₂₁₆S₁₅,the relative molecular mass was 17.06581 k D,the theoretical isoelectric point was 9.36,and it was a basic unstable protein;after IGF1 gene overexpression,the cell proliferation-related genes The expression of cyclin A2,cyclin B1 and PCNA was significantly higher than that of the control group after IGF1 gene overexpression（P<0.01）,while the expression of cyclin A2,cyclin B1 and PCNA was significantly lower after IGF1 gene silencing（P<0.01）.The CCK8 results showed that after the overexpression of the IGF1 gene,the proliferation ability of the cells was enhanced;After the IGF1 gene was silenced,the proliferation ability in the cells was weakened;transfection of overexpressed myoblasts significantly enhanced the migratory activity and the cell migration rate was highly significantly increased（P<0.01）,in contrast,the migratory activity of interfering myoblasts was significantly di minished and the cell migration rate was highly significantly decreased（P<0.01）.Overexpression of IGF1 gene expression significantly decreased the number of cells in G1 phase（P<0.05）and highly significantly increased the number of cells in the S phase（P<0.01）,while interference with IGF1 gene expression elevated the number of cells in the G1 phase and significantly decreased the number of cells in the S phase（P<0.05）.That is,IGF1 gene had a positive regulatory effect on myogenic cell proliferation.2.Study on the effect of MSTN on the proliferation of myogenic cells in Guanling cattle.The successful MSTN gene disruption vector was transfected into adult myoblasts for screening and identification.CCK8,qPCR,cell scoring and flow cytometry were used to investigate the effect of MSTN gene on the proliferation of adult myoblasts in Guanling cattle.The results showed that overexpression of MSTN gene in adult myoblasts significantly down-regulated the expression of cyclin A2 and cyclin D1 genes（P<0.01）,while silencing of MSTN gene significantly up-regulated the expression of cyclin A2 and cyclin D1 genes（P<0.01）.CCK8 results show that overexpression of MSTN gene inhibits myoblast proliferation,while interfering with MSTN gene promotes myoblast proliferation.Overexpression of MSTN gene down-regulated myogenic cell migration rate（P<0.01）,while silencing of MSTN gene up-regulated myogenic cell migration rate（P<0.01）.Overexpression of MSTN gene in myogenic cells significantly reduced the number of cells in S-phase of cell cycle（P<0.01）,while silencing of MSTN gene increased the number of S-phase cells.That is,the MSTN gene has a suppressive effect on the proliferation of adult myoblasts.3.Study on the effect of IGF1 and MSTN on the proliferation of myogenic cells in Guanling bovineIGF1 and MSTN gene overexpression vectors and interference vectors were cotransfected by liposomes,respectively,and the effects of IGF1 and MSTN gene co-interaction on myogenic cell proliferation were exa mined by CCK8,qPCR technique,cell scratching and flow cytometry.The results showed that co-expression of IGF1 and MSTN genes highly significantly down-regulated the expression of Cyclin A2 and Cyclin B1,genes related to cell proliferation（P<0.01）,while co-interference up-regulated the expression of Cyclin A2 and Cyclin B1 genes.The CCK8 results showed that co-expression inhibits myoblast proliferation,while co-silence promotes myoblast proliferation.Co-expression significantly down-regulated myogenic cell migration activity（P<0.01）,while co-interference significantly up-regulated myogenic cell migration rate（P<0.01）.Co-expression significantly reduced the number of pericellular S-phase cells,while co-silencing significantly increased the number of S-phase cells（P<0.01）,i.e.,the MSTN gene regulates IGF1 gene-mediated myogenic cell proliferation in Guanling bovine myogenic cells.In conclusion,IGF1 gene has a positive regulatory effect on myogenic cell proliferation,in contrast to MSTN gene which has a negative regulatory effect on myogenic cell proliferation there is a regulatory effect,while IGF1 and MSTN interact together to regulate myogenic cell proliferation process,MSTN gene regulates IGF1 gene mediated myogenic cell proliferation.