Preliminary Study On The Mechanism Of Estrogen Regulating Pokemon Expression In Breast Cancer

Objective:To clarify the effect of 17-βestradiol（E2）on the expression of Pokemon in breast cancer cells,and to explore the molecular mechanism of ERαregulating Pokemon expression.Methods:The experiment was divided into five parts:1 The breast cancer cells MCF-7 were used as the research object.Different concentrations(10^-9,10^-8,10^-7,10^-6 mol/L)E2 were added to stimulate for 4h,the changes of ERαand Pokemon proteins were analyzed by Western Blot.Subsequently,MCF-7 cells were stimulated with 10^-8 mol/L E2 for 1h,2h and 4h.The protein and mRNA levels of ERα,Pokemon in MCF-7 cells were analyzed by immunoblotting and real-time quantitative reverse transcription polymerase chain reaction（qRT-PCR）.2 MCF-7 cells were transiently transfected with ERαsiRNA-1,ERαsiRNA-2 and ERαsiRNA-3 respectively.The silencing effect of ERαsiRNA was detected by immunoblotting and ERα-specific siRNA was screened.3 MCF-7 cells were transiently transfected with ERαsiRNA-3,and the change of Pokemon protein was analyzed by immunoblotting.4 MDA-MB-231cells were transiently transfected with ERαplasmid,and the change of Pokemon protein was analyzed by immunoblotting.5 The Pokemon promoter plasmid and ERαsiRNA-3 were transiently co-transformed into MCF-7 cells,and the effect of ERαsiRNA-3 on the activity of Pokemon promoter was analyzed by double luciferase reporter gene assay.The Pokemon promoter plasmid and the ERαplasmid were transiently co-transformed into MDA-MB-231 cells,and the effect of the ERαplasmid on the activity of the Pokemon promoter was analyzed using a dual luciferase reporter gene assay.Results:1.Western Blot assay confirmed that the addition of 10^-8,10^-7 mol/L E2 stimulated MCF-7 cells for 4h,and the levels of ERαand Pokemon proteins decreased compared with the control group.The MCF-7 cells were stimulated with 10^-8 mol/L E2 for 1h,2h and 4h.The expression of ERα,Pokemon protein and mRNA was decreased by Western Blot and qRT-PCR after 2h and 4h stimulation.2.In MCF-7 cells,ERαsiRNA-3 has a good ERαsilencing effect.3.After silencing ERαin MCF-7 cells,Western Blot detected a decrease in the level of Pokemon protein.4.After MDA-MB-231 cells overexpressed ERα,the level of Pokemon protein increased.5.By double luciferase reporter gene activity experiments,it was found that silencing ERαcan inhibit the expression of Pokemon promoter in MCF-7 cells,and overexpression of ERαcan increase the expression of Pokemon promoter in MDA-MB-231 cells.Conclusion:E2 inhibits Pokemon mRNA transcription and protein expression in breast cancer cells,and its mechanism is related to the regulation of Pokemon promoter activity by ERα...