A Study On The Interaction Of PRMT2 And ER-α36 In Breast Cancer Cells

Objective: To construct the plasmids pYr-ads-4-ER-α36、pYr-ads-4-PRMT2、pcDNA3.1-Myc-His-ER-α36 and pcDNA3.1-PRMT2-flag.To explore the interaction between PRMT2 and ER-α36 in breast cancer cells,and to provide new insights into therapy of drug resistant breast cancer.Method: T47 D cells、MCF-7 cells、Hela cells and MDA-MB-231 cells cultured in vitro were utilized in the present study to investigate the interaction of PRMT2 and ER-α36 in breast cancer cells.Our experiments included four parts:(1)Construction of the plasmids pYr-ads-4-ER-α36、pYr-ads-4-PRMT2、pcDNA3.1-Myc-His-ER-α36 and pcDNA3.1-PRMT2-flag.(2)The levels of PRMT2 and ER-α36 in Hela cells and MDAMB-231 cells infected with pYr-ads-4-PRMT2 and pYr-ads-4 –ER-α36 were detected by real-time fluorescence quantitative PCR and Western blotting.(3)The interaction between PRMT2 and ER-α36 in the MCF-7 cells and MDA-MB-231 cells transfected the plasmids pcDNA3.1-Myc-His-ER-α36 and pcDNA3.1-PRMT2-flag were verified by CoIP.(4)The plasmids pcDNA3.1-Myc-His-ER-α36 and pcDNA3.1-PRMT2-flag was transfected in the T47 D cells and MDA-MB-231 cells to reveal the subcellular localization of PRMT2 and ER-α36 through immunofluorescence.Result:1.The pYr-ads-4-ER-α36,pYr-ads-4-PRMT2,pcDNA3.1-Myc-His-ER-α36,pcDNA3.1-PRMT2-flag were sequenced and its constructed sequence was identical with the target gene which suggested that pYr-ads-4-ER-α36、pYr-ads-4-PRMT2、pcDNA3.1-Myc-His-ER-α36、pcDNA3.1-PRMT2-flag was confirmed effectively.2.qPCR and Western blot revealed that after infecting with pYr-ads-4-ER-α36、pYr-ads-4-PRMT2,overexpression of PRMT2 and ER-α36 were observed in Hela cells and MDA-MB-231 cells.Thus the efficiency of pYr-ads-4-ER-α36 and pYr-ads-4-PRMT2 were further confirmed.3.CoIP revealed that PRMT2 interacted with ER-a36 in MCF-7 cells and MDA-MB-231 cells which was transfected pcDNA3.1-MycHis-ER-α36、pcDNA3.1-PRMT2-flag.4.Immunofluorescence showed PRMT2 was expressed in both nuclei and cytoplasm of T47 D cells and MDA-MB-231 cells which were transfected pcDNA3.1-Myc-His-ER-α36、pcDNA3.1-PRMT2-flag,and ER-α36 was expressed mostly in cytoplasm,while their fluorescence signals were overlapped.So it revealed that PRMT2 and ER-α36 were co-localized in the breast cancer T47 D cells and MDA-MB-231 cells.Conclusion:1.pYr-ads-4-ER-α36、pYr-ads-4-PRMT2、pcDNA3.1-Myc-His-ER-α36 and pcDNA3.1-PRMT2-flag was constructed successfully.2.PRMT2 interacted with ER-α36 in MCF-7 cells and MDA-MB-231 cells..3.PRMT2 was co-localized with ER-α36 in MDA-MB-231 cells and T47 D cells.