Regulation Of Oxymatrine On The Proliferation,Apoptosis And Migration Of Human Hepatoblostnoma Cell Line HepG2

Objective: To investigate the relationship between oxymatrine(OMT)and HCV NS5 A trans-regulated protein 13(NS5ATP13),and its effect and mechanism on proliferation and apoptosis in HepG2 cells.Method:1.Observe and explore the effect of OMT on proliferation and apoptosis in HepG2 cells:(1)Different concentrations of OMT were added to HepG2 cells.After interfering with HepG2 cells for 24 hours,the cellular activity was screened for the optimal OMT drug concentration in subsequent experiments by CCK-8.(2)The changes of cell viability,healing rate,migration and invasion degree and caspase-3/7 expression level were detected by adding OMT to HepG2 cells,inorder to analyzed the effect of the proliferation and migration.(3)Western blot was used to analyze the effect of OMT on the expression of apoptosis-related genes,such as Bcl-2,Bax,N-Cadherin,in order to initially explorate of the mechanism of OMT action.(4)The effect of OMT on NS5ATP13 gene expression was analyzed by real-time PCR and Western blot at the level of mRNA and protein respectively.2.Observe and explore the effect of NS5ATP13 on proliferation and apoptosis in HepG2 cells:(1)Establish an in vitro cell model of NS5ATP13 gene overexpression and interference in hepatocellular carcinoma cell line HepG2 by transient transfection of NS5ATP13 overexpression vector(pNS5ATP13)and small interfering RNA(siNS5ATP13)and their respective negative controls in HepG2 cells,and verifying it at the mRNA level.(2)Under the premise of successful verification,detect the changes of cell viability,healing rate,migration and invasion degree,Caspase-3/7 expression level,and analyze the effect of NS5ATP13 on proliferation and migration of HepG2 cells.(3)Western blot was used to analyze the effect of NS5ATP13 on the expression of apoptosis-related genes,such as Bcl-2,Bax,N-Cadherin,in order to initially explorate of the mechanism of NS5ATP13 action.3.Observe and explore the relationship between OMT and NS5ATP13 and its effect on proliferation and apoptosis in HepG2 cells:(1)To further understand the relationship between OMT and NS5ATP13,OMT was added after silencing NS5ATP13 in HepG2 cells.(2)Detect the changes of cell viability,healing rate,migration and invasion degree,Caspase-3/7 expression level,and analyze the effect of OMT on the proliferation of HepG2 cells after interfering with NS5ATP13.(3)Western blot was used to further analyze the relationship between OMT and NS5ATP13 at protein level and the mechanism of their effects on proliferation and apoptosis of hepatocellular carcinoma cell line HepG2.4.Observe and explore the relationship between OMT,NS5ATP13 and AKT/GSK/mTOR signaling pathway: Western blot was used to analyze the the expression of proteins,such as AKT,p-AKT,GSK3β,p-GSK3β(Ser9),mTOR,p-mTOR,when dealing with OMT,N5STP13 or adding OMT after silencing NS5ATP13,in order to analyze the relationship between OMT,NS5ATP13 and AKT/GSK/mTOR signaling pathway.Result:1.OMT can inhibit the proliferation and migration of HepG2 cells,promote cell apoptosis.2.Overexpression of NS5ATP13 can promote the proliferation and migration of HepG2 cells,and silencing NS5ATP13 can promote the apoptosis of HepG2 cells.3.Silencing NS5ATP13 and adding OMT can further reduce the expression of NS5ATP13 and significantly inhibit the proliferation and migration of HepG2 cells and induce apoptosis.4.Silencing NS5ATP13 and adding OMT significantly inhibited the AKT/GSK/mTOR signaling pathway.Conclusion:1.OMT and NS5ATP13 have significant effects on the proliferation and apoptosis of HepG2 cells.The mechanism may be related to the regulation of apoptosis-related gene expression in HepG2 cells,affecting the AKT/GSK/mTOR signaling pathway,and affecting the mitochondrial apoptosis pathway and EMT activation.2.OMT can inhibit the expression of NS5ATP13,and it can further reduce the expression of NS5ATP13 and increase the inhibition of HepG2 cell proliferation and migration when adding OMT after silencing NS5ATP13,indicating that NS5ATP13 may be one of the targets of OMT.