The Study Of Proliferative,Apoptosis And Molecular Mechanism After GIGYF1 Knockdown In H9C2 Myocardial Cell

Objective:In the present study,we intended to investigate the effects and mechanism of apoptosis,proliferation and differentiation by siRNA silencing GIGYF1 gene expression in H9C2 myocardial cell and provide theoretical basis for early clinical screening of congenital heart disease.Methods:H9C2 myocardial cell was transfected with three pairs of siRNA for GIGYF1 gene by liposome transfection.GIGYF1 mRNA and protein expression were detected by qRT-PCR and western blotting after siRNA transfection to find out the best method and siRNA of transfecting.The effects on the cells’ apoptosis and growth were determined by flow cytometry flow and cells colony.RT-PCR was used to detect the relative expression of GATA4,Nkx2.5 and Tbx5,which was explored the change of the cell differentiation.The mechanism of GIGYF1 knockout H9C2 myocardial cell proliferation and differentiation were analyzed by detected the protein expression of p38,P-EIF4E,P-MNK1 with western blotting.In addition,the mechanism of apoptosis in GIGYF1 knockout H9C2 myocardial cell was analyzed by detected the protein expression of P-AKT,p53,Bax and caspase3 with western blotting.Results:Transfection of two pairs siRNA can result in the mRNA and protein low expression of GIGYF1(P<0.01).The apoptosis rate of the transfection group of siRNA1869 and siRNA556 was significant higher than the transfection group of FAM-siRNA and the cell nucleus of the transfection group of siRNA 1869 and siRNA556 was significantly agglutinate and destructive.The number of cells cloned in the transfection group of siRNA 1869 and siRNA556 was significant lower than the transfection group of FAM-siRNA(P<0.01).The relative expression of GATA4 of the transfection group of siRNA 1869 and siRNA556 was significantly reduced than the transfection group of FAM-siRNA and blank group after transfection(P<0.01).The protein expression of P-AKT,p38,P-MNK1 and P-EIF4E in the transfection group of siRNA 1869 and siRNA556 were significantly reduced than the blank group after transfection(P<0.01),while the protein expression of p53,Bax,caspase3 in the transfection group of siRNA 1869 and siRNA556 were significantly higher than the blank group after transfection(P<0.01).Conclusions:(1)The sequences of siRNA1869 and siRNA556 to silence GIGYF1 gene were achieved.(2)The apoptosis,proliferation and differentiation of H9C2 myocardial cell were significantly affected after GIGYF1 siRNA transfection.The mechanism of apoptosis may be that the decreased expression of P-AKT and upregulation of p53,Bax and caspase3 lead to the apoptosis in H9C2 myocardial cell.(3)The reduction of proliferative activity and differentiation of H9C2 myocardial cell after transfection may be related to the decrease of p38,P-EIF4E,P-MNK1 and GATA4 expression.