Study On ERα Transcription Activity Effected By PRMT2 And Its Spliceosome

Objective: Compared the effects of protein arginine methyltransferαse 2 (PRMT2) and its novel variants PRMT2α,PRMT2β,PRMT2γon the transcriptional activity of ERαunder different conditions. And to observe the intERαction between ERαand PRMT2 or its Splicings PRMT2α,PRMT2β,PRMT2γin vitro.Methods: Extracted pcDNA3.0-PRMT2,pcDNA3.0-PRMT2α,pcDNA3.0 -PRMT2β,pcDNA-PRMT2γand pcDNA3.0 empty vector plasmid respectively and then cotranfected them with the pcDNA3.0-ERαinto 293T cells (using liposomes-mediated) . Including choice of consensus sequences regulated by the ERE luciferαse gene (ERE-Luc) as reported gene which was detected by fluorescence detector under different reaction conditions and then compared PRMT2 and its spliceosome PRMT2α,PRMT2β,PRMT2γof ERαtranscriptional activity.This part consisted of 2 components, one was compared the effects of PRMT2 or its variants PRMT2α,PRMT2β,PRMT2γon the transcriptional activity of ERαin the absence and presence of estrogen. The second was observed the impact of PRMT2 and its spliceosome PRMT2α, PRMT2β, PRMT2γon ERαtranscriptional activity under the inclusion of estrogen and estrogen inhibitor (ICI182,780 , 4-OHT) .The recombinants which expressed the GST alone and GST-PRMT2 or its spliceosome were transformed into E.coli.BL21.The interesting protein were inspected by SDS-PAGE and coomassie brilliant blue stainging.The fusion protein and GST was purified by affinity chromatography column. Using GST pull-down were detected PRMT2 and its spliceosome interαct with ERαin vitro and its correlation with the estrogen.Result: Under using dual-lucifERαse reporter assay system, PRMT2 and its variants could not enhance the transcriptional activity of ERαwhen estrogen was absence. Compared with empty vector group, the ERαtranscriptional activity of PRMT2 and its variants enhanced the ERαtranscriptional activity about 2.9 fold,1.7 fold,1.5 fold and 2.1 fold respectively. Comparison of estrogen and estrogen inhibitors, there were circumstances which under the presence of estrogen inhibitors that ERαtranscriptional activity is lower . In GST pull-down, it showed that in absence and presence of estrogen, the GST- PRMT2α,GST-PRMT2β,GST-PRMT2γfusion protein could separately combine with ERα.Conclusion: PRMT2α,PRMT2β,PRMT2γcould ehance the transcription activity of ERαin a hormone-dependent as PRMT2 and the transcription activity decreased in the presence of estrogen inhibitors. The novel variants PRMT2α,PRMT2β,PRMT2γcould combine with ERαnever depended on the presence of estrogen in vitro, but estrogen may could enhance the effect of it. PRMT2α,PRMT2β,PRMT2γmay also be the coactivators of ERα, as same as PRMT2 through ERαsignaling pathway. It may play important roles in the occurrence and development of breast cancer and was expected to become a new target for breast cancer treatment.