Expression Of Nrf3 In Colorectal Cancer And Its Antioxidant Effect And Mechanism

Objective:To investigate the expression of Nuclear factor E2 related factor 3(Nrf3)in colorectal cancer and the effect of overexpression of Nrf3 on antioxidant activity of colorectal cancer cells.Methods:1.TCGA database and immunohistochemistry were used to detect the expression of Nrf3 at transcriptional level and protein level in the tissues of clinical colorectal cancer patients.2.Nrf3 protein levels were measured by Western blot after HCT116,SW620 and HT29 cells were treated with Hydrogen peroxide(H2O2)of different concentrations(0,200,400,600 μM))for 12 hours.3.A stable Nrf3 overexpression cell line(OE-Nrf3-HCT116)was constructed,and the expression levels of Nrf3 mRNA and protein were detected by qPCR and Western blot.4.After distinct concentrations(0,200,400,600 μM)of H2O2 treated colorectal cancer cells,Nrf3 protein level was checked by Western blot,and cell inhibition rate was tested by CCK-8,the changes of intracellular ROS level and apoptosis were checked by flow cytometry and fluorescence microscope,the content of malondialdehyde(MDA)in cells was detected by enzyme labeling instrument.5.Western blot was used to detect the protein levels of Nrf3,P-AKT,AKT,Bcl-2,P-JNK,P-JNK,P-p38 and P38 in Control cells treated with 400 for(0,3,6,12 h).6.Western blot was used to detect the expression of Nrf3,PI3K,P-AKT,AKT,Bcl-2,P-JNK,JNK,P-p38 and P38 in Control and OE-Nrf3-HCT116 cells.7.After treating the Control and OE-Nrf3-HCT116 groups with H2O2 at different concentrations(0,200,400,600 μM)for 12 h and Control and OE-Nrf3-HCT116 groups at different times(0,3,6,12 h)with 400 μM concentrations,Western blot detected the Nrf3,P-AKT,AKT,Bcl-2,P-JNK,JNK,P-p38,p38 protein levels of the cells.8.CCK-8 and flow cytometry were used to detect diverse concentrations of 5-Fluorouracil(5-FU)(0,1.5,3,6,12,24 μM)about the inhibition rate and apoptosis of Control and OE-Nrf3-HCT116 cells.Results:1.The expression of Nrf3 in tissues of colorectal cancer was significantly ascended in transcriptional level and protein levels.2.Western blot detection showed that the expression of Nrf3 increased with diverse concentrations of H2O2(0,200,400,600 μM)after treating HCT116,SW620and HT29 cells for 12h.3.The mRNA and protein expression levels of Nrf3 in OE-Nrf3-HCT116 cells were significantly higher than those in the control group.4.When HCT116 and OE-Nrf3-HCT116 cells were dealed with distinct concentrations of H2O2(200,400,600 μM),the inhibition rate of control group(58.17%,83.33%,93.63%)was obvious higher than that of OE-Nrf3-HCT116 cells(23.20%,35.30%,70.30%),the ROS value of the control group(47.3%,56.4%,74.23%)was higher than that of OE-Nrf3-HCT116 cells(35.9%,45.3%,47.83%);when the concentration is 600μM,the MDA content of OE-Nrf3-HCT116(0.3025 nmol/mg.prot)was obvious lower than that of the control group(0.6922 nmol/mg.prot),the apoptosis rate of the control group(23.79%,24.7%,24.61%)was higher than that of OE-Nrf3-HCT116(12.03%,12.14%,13.18%).5.The expression levels of Nrf3 and P-AKT,P-JNK and P-p38 decreased firstly and then increased with time in Control group,Bcl-2 increased with time,but the expression levels of JNK,p38 and Akt were not significantly different in Control group.Among them,the expression levels of Nrf3 and P-AKT,P-JNK,P-p38 and Bcl-2 were upregulated at 12h hours.6.The expression levels of Nrf3,P-AKT,Bcl-2,P-JNK,P-p38 in Nrf3-HCT116 cells were higher than those in Control cells.7.The expressions of Nrf3,P-AKT,Bcl-2,P-JNK and P-p38 in OE-Nrf3-HCT116 cells treated with different H2O2 concentrations were higher than those in the control group and increased with concentrations,while the expressions of AKT,JNK and p38 proteins were not significantly different;At a concentration of 400 μM and at different times(0,3,6,12 h),the expression levels of Nrf3,P-AKT and P-JNK in Nrf3 overexpressed cells were higher than those in the control group at 0h and 12 h,and the expression levels of Nrf3 and P-AKT,P-JNK and P-p38 in the control group and experimental group were decreased first and then increased over time,and the expression of Bcl-2 protein increased over time,and there was no significant difference in the expression of JNK,p38 and AKT.8.With diverse concentrations of 5-FU(1.5,3,6,12,24μM)treated HCT116 and OE-Nrf3-HCT116 cells for 48h,the inhibition rate of control group(50.74%,61.05%,67.32%,73.94%,75.39%)was obvious higher than that of overexpressed cells(29.29%,41.61%,46.84%,55.46%,58.20%),and the apoptosis rate of the control group(55%,62.3%,68.23%)was obvious higher than that of the experimental group(22.4%,24.73%,30.1%).The difference was statistically significant(P<0.05).Conclusion:Nrf3 is up-regulated in colorectal cancer tissues compared with paracancer tissues.Nrf3 can reduce H2O2-induced oxidative stress injury of HCT116 cells,and this protective effect was related to P-JNK,P-p38,P-Akt and Bcl-2 related proteins.