The Mechanism Of ERα Expression Regulated By RNPC1, A RNA Binding Protein, In Breast Cancer

Objective: To investigate the mechanism of ERα regulated by the RNA binding protein RNPC1 and the reverse mechanism of RNPC1 regulated by ERα. To explore the biological and endocrine therapy significance of the feedback loop between RNPC1 and ERα.Materials and methods: Immunohistochemical(IHC) staining was used to detect the expression of RNPC1 and ERα in 90 breast cancer tissues. Immunofluorescence was used to clarify the celluer location of RNPC1 and ERα. The breast cancer cells(MCF-7, BT474, MDA-MB-231, SUM 1315) were stably transfected RNPC1 and ERα overexpression and knockdown lentivirus, respectively. Western blot and q RT-PCR were applied to analyze the protein and m RNA expression of RNPC1 and ERα. The RNPC1 overexpression and knockdown cells were treated with actinomycin D to detect the stability of ERα transcript. RNA immunoprecipitation(RIP) was performed to investigate the association between RNPC1 and ERα transcript. RNA electrophoretic mobility shift assay(REMSA) was performed to detect the binding site(s) of RNPC1 in ERα transcript and dual-luciferase reporter assay was used to confirm the function of the binding sites. The proliferation of RNPC1 knockdown cells treated with estrogen was assessed by cell counting kit-8(CCK-8). Western blot and q RT-PCR were applied to investigate the endogenous expression of RNPC1 and ERα in MCF-7 and BT474 treated with estrogen.Results: RNPC1 expression was obviously higher in ERα positive breast cancers compared with ERα negative breast cancers(p<0.05). RNPC1 was mainly expressedin nucleus and cytoplasm, and ERα was mainly expressed in the nucleus. ERα expression was increased by ectopic overexpression of RNPC1 in MCF-7 and BT474 cells, while RNPC1 down-regulation decreased ERα expression. RNPC1 showed no influence on the ERβ expression in breast cancer cells. In ER negative cells MDA-MB-231 and SUM 1315, there was no change of ERα expression after RNPC1 overexpression or knock down. RNPC1 overexpression increased the stability of ERα m RNA, while knockdown decreased. RNPC1 could bind to ERα 3’ untranslated regions which contain multiple AU/U-rich elements. The overexpression of ERα could reversely reduce RNPC1 expression in breast cancer cells. The expression of RNPC1 was increased after ERα knockdown. Estrogen could increase the proliferation of RNPC1 knockdown cells and reduce the endogenous expression of RNPC1.Conclusions: RNPC1 expression was significantly correlated with ERα in breast cancer. RNPC1 was mainly expressed in nucleus and cytoplasm, and ERα was mainly expressed in the nucleus. ERα expression was increased by ectopic overexpression of RNPC1, while RNPC1 knockdown decreased ERα expression. RNPC1 could bind to ERα 3’ untranslated regions which contain multiple AU/U-rich elements and increase its stability. ERα could reversely down-regulate endogenous RNPC1 expression. Estrogen could reduce the endogenous expression of RNPC1. The feedback loop between RNPC1 and ERα might provide highlights for clinical endocrine-therapy and research.