Effects Of Oxymatrine On Biological Characteristics And Epithelial Mesenchymal Transition Of Human Hepatocellular Carcinoma HepG2 Cells Induced By TGF-β1 And Its Mechanism

Objective: To explore the effect of oxymatrine on the biological characteristics and epithelial mesenchymal transition of human hepatoma HepG2 cells induced by TGF-β1 and its possible mechanism.Methods:(1)the EMT model of human hepatoma HepG2 cells was established by inducing epithelial mesenchymal transition(EMT)of human hepatoma HepG2 cells with TGF-β1.The experiment was divided into blank group,model group,oxymatrine 1 mg/ml group,oxymatrine 2 mg/ ml group and oxymatrine 4 mg/ml group.(2)Cell couting kit 8method was used to detect the inhibitory effect of oxymatrine on the proliferation of HepG2 cells.(3)The effect of oxymatrine on apoptosis of HepG2 cells was detected by flow cytometry.(4)The effect of oxymatrine on cell cycle of HepG2 cells was detected by flow cytometry PI/RNA single staining.(5)The migration ability of HepG2 cells was detected by scratch test.(6)The invasion ability of HepG2 cells was detected by Transwell chamber method.(7)Real time quantitative polymerase chain reaction(q RT-PCR)was used to detect the expression levels of mir-204 and SIRT1 in HepG2 cells.(8)Western blot was used to detect the expression of E-cadherin,Vimentin,snail 1,Twist1 and ZEB1 in HepG2 cells.The changes of E-cadherin,vimentin,snail 1,Twist1 and ZEB1 before and after treatment with oxymatrine were compared.(9)Correlation analysis was used to determine the correlation between miR-204 and HepG2 cell inhibition rate,apoptosis rate,E-cadherin and Vimentin expression.Results:(1)CCK8 test results showed that oxymatrine inhibited the proliferation of HepG2 cells which had EMT effect.With the increase of oxymatrine concentration,the inhibition rate of hcg2 on the human hepatocarcinoma cells with EMT increased,and the inhibition rate between groups was different(P<0.05).(2)The results of flow cytometry showed that there was no significant difference in the apoptosis rate between the blank group and the model group(P>0.05);Compared with the model group,the apoptotic rates of HepG2 cells in the three groups were increased(P<0.05),and the apoptotic rates among the three groups increased with the increase of the concentration of oxymatrine(P<0.05).The results of flow cytometry showed that the distribution of HepG2 cells in G0/G1 phase and S phase was significantly different among the three groups(P<0.05).Compared with the model group,the proportion of HepG2 cells in G0/G1 stage was increased in the Oxymatrine treated group(P<0.05).After treatment with matrine,the S stage distribution rate of cells in each group decreased(P<0.05),but there was no significant difference in G2/M phase distribution rate among each group(F=3.190,P>0.05).(3)The results of scratch test showed that the difference of wound healing rate between groups was statistically significant(F=17.007,P<0.001).The migration ability of HepG2 cells in model group was higher than that in the blank group(P<0.05);Compared with the model group,the migration capacity of HepG2 cells in OM 2 mg/ml group and OM 4 mg/ml group decreased(P<0.05),suggesting that after oxymatrine,the migration ability of HepG2 cells decreased and showed dose-dependent effect.(4)The results of Transwell chamber test showed that there was significant difference in the number of cell invasion between the groups(F=735.245,P<0.001).Compared with the blank group,the number of cells in the model group increased significantly(P<0.001).Compared with the model group,the number of HepG2 cells in the three groups after oxymatrine action was significantly reduced(P<0.001),and the difference between the three groups was statistically significant(P<0.001).That is,after the oxymatrine action,the invasion ability of HepG2 cells in each group decreased,and the invasion capacity of HepG2 cells was dose-dependent.(5)The results of q RT-PCR showed that there was a significant difference in the relative expression of miR-204 among the groups(F=5.348,P<0.05).The expression of miR-204 in HepG2 cells of model group and oxymatrine group was higher than that in the blank group(P<0.05);Compared with the model group,the miR-204 expression level in HepG2 cells of the three groups after oxymatrine action increased(P<0.05),which proved that oxymatrine could increase the expression level of miR-204 in HepG2 cells,and it was dose-dependent.However,there was no significant difference in SIRT1 expression between groups,suggesting that oxymatrine may not have significant effect on SIRT1.(6)The results of Western blot showed that there was significant difference in E-cadherin expression among the groups(F=4.249,P<0.05).Compared with the blank group,the expression level of E-cadherin in HepG2 cells decreased(P<0.05),and the Vimentin expression level was increased(P<0.05);Compared with the model group,the expression level of E-cadherin in HepG2 cells of the three groups increased(P<0.05),and the expression level of Vimentin decreased(P<0.05),suggesting that oxymatrine could up regulate the expression of E-cadherin and down regulate the expression of Vimentin,and its effect on both groups was dose dependent.The expression of Snail1,Twist1 and ZEB1 in each group was significantly different(F=50.935,P<0.001).Compared with the control group,the expression of Snail1 in model group was significantly increased(P<0.001),and that of ZEB1 and Twist1(P<0.05).Compared with the model group,the relative expression of Snail1 and ZEB1 in OM 2 mg/ml group and OM 4 mg/ml group were significantly lower(P<0.001),and the relative expression of Twist1 was decreased(P<0.05),and the Snail1 and the Snail1 of HepG2 cells were decreased after the action of oxymatrine The expression of Snail1,Twist 1 and ZEB1 in HepG2 cells was down regulated when the concentration of oxymatrine was more than 2mg/ml after the treatment.(7)The results of univariate correlation and regression analysis showed that the expression of miR-204 in HepG2 cells was positively correlated with the expression of E-cadherin(r=0.767,P<0.05),and negatively correlated with Vimentin expression(r=-0.765,P<0.05);The expression of miR-204 in HepG2 cells was positively correlated with the apoptosis rate(F=12.272,P<0.05).Conclusion:1.Oxymatrine can up regulate the expression of miR-204,and affect the proliferation and cell cycle of HepG2 cell EMT model,arrest the cell division and promote apoptosis.2.Oxymatrine can weaken the EMT effect of HepG2 cells and inhibit the migration and invasion of EMT model of HepG2 cells.The mechanism of action may be that Oxymatrine up regulates the expression of miR-204 and down regulates the expression of Snai1,Twist1 and ZEB1.3.This study found that oxymatrine had no obvious effect on SIRT1 gene.