Role Of CYP2E1 And PI3K/Akt/mTOR Signaling Pathway In Gastric Cancer

Objective:1.The expression of CYP2E1 in poorly differentiated human gastric cancer cell lines and normal gastric mucosal epithelial cell line was compared and analyzed to verify the activation of CYP2E1 in human gastric cancer.To search the biomarkers of gastric cancer.2.The gastric cancer which is overexpression of CYP2E1 was constructed,in order to investigate the effects of CYP2E1 on proliferation,cell cycle,apoptosis,invasion and migration of gastric cancer cell with over-expression of CYP2E1.3.The aim of this study was to observe the effect of CYP2E1 on the key genes of PI3K/Akt/mTOR signaling pathway in human gastric cancer cell and to explore the possible molecular mechanism of CYP2E1 regulating the growth of gastric cancer cells.4.The aim was to observe the effect of PI3K inhibitor LY294002,mTOR inhibitor Rapamycin on the expression of CYP2E1 in gastric cancer cells,and to propose a scientific hypothesis on the mechanism between CYP2E1 and PI3K/Akt/mTOR pathway in gastric cancer.To explore the molecular mechanism of PI3K/Akt/mTOR regulating CYP2E1 in gastric cancer cells in order to provide more experimental basis for clinical application of CYP2E1.Method:1.Human gastric cancer cell lines MGC-803,SGC-7901,BGC-823 and normal gastric epithelial cell line GES-1 were cultured in vitro.Real-time fluorescence quantitative PCR was used to detect the mRNA expression of CYP2E1;western blot method was used to detect the quantitative expression of CYP2E1 and ACTIN protein,Western Blot assay was used to detect the expression of CYP2E1 protein and ACTIN protein.Semi-quantitative analysis was performed with CYP2E1/ACTIN gray scale ratio.2.The construction of CYP2E1 high expression lentivirus vector and its corresponding empty vector was completed by Shanghai JiKai Gene Company.Lentivirus-mediated over-expression of CYP2E1 in MGC-803 cell lines was established.MGC-803 cell group was set as control.To construct lentiviral vector mediated overexpression of CYP2E1 in MGC-803cell group and to transfect corresponding lentivirus empty vector MGC-803 cell group.CYP2E1 lentiviral vector and lentiviral empty vector were transfected into MGC-803 cells.Fluorescence was observed under a fluorescence inverted microscope.After screening with puromycin,total RNA was extracted from MGC-803 cell group,MGC-803 NC cell group?lentivirus empty vector transfected MGC-803 group?and MGC-803 CYP2E1 cell group?CYP2E1 overexpressing MGC-803 cell line group?.Real-time quantitative PCR and Western Blot were used to verify the successful construction of MGC-803 cell line group with over-expression of CYP2E1 at mRNA and protein levels,respectively.3.To verify the effect of overexpression of CYP2E1 on the biological function of MGC-803 cellsMGC-803 CYP2E1 cell group was used as experimental group,and MGC-803 NC cell group was used as control.CCK-8 method was used to test the proliferation ability of MGC-803 cells overexpressing CYP2E1 at different time gradients and draw growth curve;Annexin V-APC/PI staining combined with flow cytometry was used to detect the apoptotic rate of MGC-803 cells overexpressing CYP2E1;flow cytometry was used to determine the cell cycle change of MGC-803 cells overexpressing CYP2E1;CYP2E1 overexpressing MGC-803cells were collected and planked into Transwell chamber and the invasion and migration ability of gastric cancer cells were detected by the upper chamber.4.The expression of PI3K/Akt/mTOR signaling pathway PI3K,Akt and mTOR genes in MGC-803 cells with overexpression of CYP2E1 was detected by real-time quantitative PCR.The total protein of PI3K,Akt,mTOR^Ser2448 and phosphorylated protein p-PI3K,p-Akt,p-mTOR^Ser2448,p-4EBP1,p-P70S6K^Ser371,p-P70S6K^Thr389 and ACTIN protein were detected by Western Blot method.Semi-quantitative analysis was performed with GENE/ACTIN gray scale ratio.5.According to the experimental results of inhibiting the proliferation of gastric cancer cell line by PI3K inhibitor LY294002,three drug concentration points were selected to intervene in gastric cancer cell lines MGC-803 and SGC-7901 with LY294002.Western Blot was used to detect the protein expression of CYP2E1 in MGC-803 and SGC-7901 cells of gastric cancer.6.According to the results of inhibition of proliferation of gastric cancer cells by mTOR inhibitor Rapamycin,three drug concentration points were selected to intervene in gastric cancer cell lines MGC-803 and SGC-7901 with Rapamycin.Western Blot was used to detect the expression of CYP2E1 in gastric cancer MGC-803 cells by Rapamycin.Result:1.The mRNA expression level of CYP2E1 in normal gastric epithelial cells GES-1 was significantly higher than that in gastric cancer cells MGC-803,SGC-7901 and BGC-823?P<0.05?.Western Blot results showed that the expression of CYP2E1 protein in gastric cancer cell lines SGC-7901 and BGC-823 was significantly higher than that in normal gastric epithelial cells GES-1?P<0.05?.There was no significant difference in the expression of CYP2E1 between gastric cancer MGC-803 and normal gastric epithelial cells GES-1?P>0.05?.2.Construction and verify the overexpressing CYP2E1 of MGC-803 cell lineCYP2E1 lentivirus vector and lentivirus empty vector were transfected into MGC-803cells respectively.The mRNA expression of CYP2E1 in MGC-803 cell group was not significantly different from that in MGC-803 NC cell group?P>0.05?.The mRNA expression of CYP2E1 in MGC-803 CYP2E1 cell group was significantly higher than that in MGC-803cell group?P<0.05?and MGC-803 NC cell group?P<0.05?..There was no difference in the protein expression of CYP2E1 between MGC-803 cell group and MGC-803 NC cell group?P>0.05?.Compared with MGC-803 cell group and MGC-803 NC cell group,the protein expression of CYP2E1 protein in MGC-803 CYP2E1 cell group was significantly increased?P<0.05?.3.Effects of overexpression of CYP2E1 on proliferation,cycle,apoptosis,invasion and migration of gastric cancer MGC-803 cellsCCK-8 assay showed that the Growth Curve of MGC-803 CYP2E1 cell group was higher than that of MGC-803 NC cell group at different time points,and the difference between groups was statistically significant?P<0.05?.Flow cytometry showed that the cell cycle distribution in G0/G1 phase increased in MGC-803 CYP2E1 group?P<0.05?,and decreased in G2/M phase?P<0.05?.The cell cycle distribution in S phase decreased,but the difference was not statistically significant?P>0.05?.Annexin V-APC/PI staining combined with flow cytometry showed that the a poptosis rate of MGC-803 NC group was significantly higher than that of MGC-803 CYP2E1 group?P<0.05?.Transwell assay showed that the number of cell migration and invasiveness in MGC-803CYP2E1 group were both significantly higher than that in MGC-803 NC group?P<0.05?.4.Changes in the expression of key genes in PI3K/Akt/mTOR signaling pathway in MGC-803 cells with overexpression of CYP2E1Real-time quantitative PCR results showed that the difference was not statistically significant in PI3K and Akt gene expression between MGC-803 CYP2E1 cell group and control MGC-803 NC cell group?P>0.05?.The expression of mTOR gene in MGC-803CYP2E1 cell group was higher than that in control group?P<0.05?.Western blot results showed that the expression of PI3K,p-Akt,p-mTOR,p-P70S6K^Ser371protein in MGC-803 CYP2E1 cell group was higher than that in control group;the expression of p-PI3K,Akt,mTOR,p-P70S6K^Thr389 protein was unchanged;the expression of p-4EBP1protein was decreased.5.Effect of LY294002,a targeted inhibitor of PI3K/Akt/mTOR signaling pathway,on the protein expression of CYP2E1 in gastric cancer MGC-803Western blot was used to detect the expression of CYP2E1 protein in gastric cancer MGC-803 and SGC-7901 that were treated with LY294002 for 24 hours.Compared with the control group,the expression of CYP2E1 in 12.5,25,50?mol/L concentration group was lower than that in the control group.6.Effect of Rapamycin,a targeted inhibitor of PI3K/Akt/mTOR signaling pathway,on the protein expression of CYP2E1 in gastric cancer MGC-803 and SGC-7901 cell linesWestern blot was used to detect the expression of CYP2E1 protein in gastric cancer MGC-803 and SGC-7901 that were treated with Rapamycin for 24 hours.Compared with the control group,there was no significant difference in CYP2E1 expression in Rapamycin concentration group.Conclusion:1.The transcriptional level of CYP2E1 was in the state of abnormal inactivation in gastric cancer.The mRNA level of CYP2E1 maybe the biomarker of gastric cancer.The transcriptional level of CYP2E1 is not consistent with its translation level in gastric cancer.2.CYP2E1 plays an important role in promoting the proliferation,inhibiting apoptosis,changing the cell cycle and promoting metastasis of gastric cancer.CYP2E1 is a key gene in promoting the progression of gastric cancer.3.The increase of CYP2E1 may be related to PI3K/Akt/mTOR signaling pathway,which is located in the upstream of Akt and mTOR.By regulating the phosphorylation of Akt and mTOR,the protein activation at the downstream P70S6K^Ser371 and 4EBP1 sites of PI3K/Akt/mTOR signaling pathway is regulated,thus may be the reason why CYP2E1 can affecting the biological functions of gastric cancer cells.CYP2E1 is regulated by PI3K and not by mTOR.That is,CYP2E1 may be downstream of PI3K in PI3K/Akt/mTOR signaling pathway.The expression of CYP2E1 can be regulated by PI3K inhibitor LY294002.