The Effects Of CRISPR/Cas9 Knockout Of MSTN Gene On Proliferation And Glucose Metabolism Of C2C12 Cells

As a negative regulator of skeletal muscle,Myostatin(MSTN)inhibits muscle growth.Understanding the molecular mechanisms that regulate skeletal muscle development and muscle fiber phenotypic characteristics is of great significance for breeding high-quality commercial animals.In this study,C2C12 was used as the research object to construct A C2C12 monoclonal cell line with MSTN gene knockout,and to detect the effects of MSTN gene knockout on C2C12 cell proliferation and glycometabolism-related proteins.This study provides a basis for revealing the molecular mechanism of MSTN regulating myoblast differentiation and muscle fiber types.Construction of C2C12 monoclonal cell line knocking out MSTN gene: The sg RNA-MSTN was designed and synthesized,ligated with the p X601 vector digested by Bas Ⅰ and transformed,and a single colony was picked for sequencing and identification to obtain the correct expression vector p X601-Sa Cas9-sg RNA-MSTN.The purine resistance gene was amplified from the p LKO.1 vector and inserted into the p X601 expression vector to obtain a purine-resistant eukaryotic expression plasmid.The plasmid was transfected into C2C12 cells,the resistant cells were screened with puromycin,and the monoclonal cell line was cultured by limiting dilution method.The MSTN gene knockout C2C12 monoclonal cell line was identified by Western blot and sequencing,and named for MSTN-KO.Knockout of MSTN gene promotes C1C12 cell proliferation: The cell proliferation activity and cell cycle were detected by CCK kit and flow cytometry.The results showed that the proliferation activity of MSTN-KO cells was significantly improved,the cells in G1 phase were significantly reduced,and the cells in G2 and S phases were significantly increased;RT-q PCR and Western Blot analysis showed that the expressions of cell growth factors such as Myo G,Myo D and CDK2 in MSTN-KO cells were significantly increased,and the expressions of factors such as P21 and Smad2 were significantly decreased.It is suggested that knocking out MSTN gene can improve the proliferation ability of C2C12 cells.Knockdown of MSTN gene enhances cellular glycolysis by upregulating PFKFB3 through AKT/m TOR signaling pathway: RT-q PCR analysis of the expression levels of TCA cycle rate-limiting enzymes CS,IDH,OGDC and key glycolysis enzymes HK,PK,PFK1 and PFKFB3 showed that the expression levels of TCA cycle rate-limiting enzymes in MSTN-KO cells were all the same.decreased,and the expression of key glycolysis enzymes was increased;MSTN-KO cells were treated with PFKFB3 inhibitor 3PO,and it was found that the protein expressions of PFK1 and PFKFB3 were decreased;Both flux and Fru-2,6-P2 content were increased;MSTN-KO cells were treated with AKT and m TOR inhibitors,and PFKFB3 protein expression was found to be decreased.Therefore,it is speculated that MSTN can enhance cellular glycolysis by up-regulating PFKFB3 through the AKT/m TOR pathway.