Nrf1 Activated Bcl-2 To Prevent Cellular Apoptosis To Cisplatin In MCF-7 Cells

Objective:By using cellular and molecular technologies,signaling pathways at the transcriptional level,This study aims to investigate the effects and mechanisms of Nrf1 in drug sensitivity to cisplatin in MCF-7cells.Methods:1.We constructed Nrf1 stable over expressed,and knockdown MCF-7 cells by the Lentivirus Package and Transfection system.The lentiviral vector Lv07 was used as negative control.2.To observe drug sensitivity of each cell to cisplatin,we treated Nrf1 over expressed MCF-7 cells,Nrf1 knockdown MCF-7 cells,and the control cells with differente concentrations of cisplatin for 48 h,and analysed by MTT assay.3.When the control MCF-7 cells were treated by 3μM cisplatin for 48 h,we extracted the protein and detected endogenous Nrf1 expression induced by western blotting.4.To observe the cell apoptosis,we treated Nrf1 over expressed MCF-7 cells,Nrf1 knockdown MCF-7 cells and the control cells with 3 μM cisplatin,respectively,and analyzed by TUNEL assay.Then the expression levels of anti-apoptotic gene Bcl-2 and pro-apoptotic gene Caspase-3 were detected by real-time PCR and western blotting.5.We cloned the wild type promoter of Bcl-2 and the truncted promoter that removed the ARE fragment,and use luciferase reporter gene assay to detect the effect of Nrf1 on the Bcl-2 transcriptional activity.Results:1.We constrcuted the Nrf1 stable over-expression and knockdown MCF-7 cell lines successfully.2.The MTT results showed that when control MCF-7 cells were treated with 3u M cisplatin for 48 h,MCF-7 cells were about 50 % survival.However,Nrf1 over expressed MCF-7 cells still had 72% survival,but the cell viability of Nrf13.knockdown MCF-7 cells was only 21%,significantly lower than that of the wild type cells.4.The control MCF-7 cells which treated by cisplatin has a high level on the expression of the endogenous Nrf1 than the non-treated cells.5.The results of TUNEL analysis showed that the apoptosis rate of wild-type MCF-7 cells with cisplatin treatment was 54.2%,and the Nrf1 over expressed cells was only 28.1%,while the Nrf1 knockdown cells was increased to 83%.6.Real-time PCR and western blotting show that when Nrf1 was overexpressed,the m RNA level and protein level of Bcl-2 were increased,while the m RNA level and protein level of Caspase-3 were reduced.However,when Nrf1 was knockdown,the m RNA level and protein level of Bcl-2 were decreased,and the m RNA level and protein level of caspase-3 were increased.7.The luciferase reporter gene assay showed higher transcriptional activity of Bcl-2 in Nrf1 over expressed MCF-7 cells than that of control MCF-7 cells.When we truncated this promoter region to remove the ARE fragment,the activated transcriptional activity was inhibited.Conclusions:1.Cisplatin could induced the expression of Nrf1.2.Nrf1 over expression reduce the sensitivity to cisplatin of MCF-7 cells,whereas Nrf1 knockdown can increase the sensitivity of MCF-7 cells to cisplatin,make it easier for cell death.3.Nrf1 inhibited cisplatin-induced apoptosis in MCF-7 cells.4.Nrf1 up-regulates Bcl-2,and down-regulates Caspase-3 on both m RNA level and protein level.5.The anti-apoptotic effect of Nrf1 was obtained by activating the transcriptional activity of Bcl-2 through the ARE fragment located in the promoter.