Pokemon Stabilizes Er-α By Inhibiting ER-α Ubiquitinated Degradation In Breast Cancer Cells

Objective: To study the mechanism by which transcription factor Pokemon inhibits ubiquitinayted degradation of ER-α,and to provide experimental evidence and theoretical basis for exploring new drug targets for the treatment of breast cancer.Methods: In vitro cultured ER-α positive breast cancer cell lines MCF-7,T47 D and ER-α negative breast cancer cell lines MD-MBA-231,MD-MBA-468,Hs-578 T were used as experimental subjects,the nucleic acid expression level and protein expression level of Pokemon were detected by qRT-PCR and Western blot respectively.The expression of Pokemon in MCF-7 and T47 D cells was knocked down by Pokemon siRNA,and the expression of ER-α nucleic acid and protein levels was detected.To further explore the specific mechanism of Pokemon regulation of ER-α,the effect of Pokemon on the half-life of ER-α protein was detected by Cycloheximide tracing assay.Meanwhile,proteasome inhibition MG132 was added to MCF-7 and T47 D cells knocking down Pokemon,to determine whether the degradation of ER-α can be affected by proteasomal pathway.In addition,it was confirmed by immuno-precipitation experiments whether Pokemon regulates the degradation of ER-α through the ubiquitination pathway.Finally,the key E3 ligase was screened by qRT-PCR and Western blot.Results :1)The expression of Pokemon was positively correlated with ER-α by qRT-PCR and Western blot analysis.Silencing Pokemon inhibited the expression of ER-α in MCF-7 and T47 D cells.2)The Cycloheximide tracking assay demonstrated that the half-life of ER-α degradation in MCF-7 and T47 D cells silenced by Pokemon was significantly shortened.The proteasome inhibitor MG132 was added to to MCF-7 and T47 D cells silencing Pokemon,which can successfully rescued the degradation of ER-α protein.3)the ubiquitination of 293 T cells was significantly increased after silencing Pokemon by immuno-precipitation technique.4)The nucleic acid and protein levels expression of related E3 ligase in MCF-7 and T47 D cells silenced by Pokemon was inhanced.Conclusion: Pokemon can promote the expression of ER-α in breast cancer cells by down-regulating the expression of E3 ligase and Ubiquitin-Proteasome System.