MiR-92 Is a Novel Regulator of ERβ1 Expression in Breast Cancer.

Abstract

Abstract Down-regulation of ERβ1 expression is thought to contribute to carcinogenesis in the breast and consequently is seen in many breast cancers. The molecular mechanisms responsible for the down-regulation of ERβ1 remain unclear. microRNAs are a novel family of regulators of gene expression that have been shown to act on the expression of many critical cancer genes but their relationship with ERβ1 has not so far been demonstrated. The aims of this study were to establish whether miR-92 regulates ERβ1 expression, and whether this regulation plays a role in defining ERβ1 expression levels in breast cancers.Using a bioinformatics approach we initially identified potential binding sites for miR-92 within the 3' untranslated regions of ERβ transcripts using RNAhybrid software (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/). Two conserved target-sites for miR-92 were identified within the ERβ1 3'-UTR sequence contained within Genebank. We confirmed the expression of these UTRs in MCF7 cells. Next, we performed 3' RACE reactions to determine the 3'-UTR sequence of ERβ1 in MCF7 cellsWe used qPCR analyses of expression in paired normal breast and breast tumour samples (n=6) to examine the relative expression of miR-92 and ERβ1. Upregulation of miR-92 expression was observed in breast tumours compared with normal breast. An inverse relationship with ERβ1 expression was observed in these samples. In a separate cohort of breast tumours (n=36), a significant negative correlation between ERβ1 mRNA and miR-92 was observed (Spearman's correlation coefficient, r = -0.5, p=0.001). Elevated ratios of ERβ1 mRNA /miR92 were also observed in ERβ1 positive compared to ERβ1 negative cells lines.Inhibition of miR-92 in MCF-7 cells increased ERβ1 expression in a dose–dependent manner at RNA levels. Enhanced GFP reporter constructs containing miR-92 binding sites from the 3'-UTR of ERβ1 were used to determine whether miR-92 downregulates ERβ1 via the direct targeting of this 3'-UTR. Inhibition of miR-92 increased the translational efficiency (protein produced per unit of mRNA) of the GFP reporter, confirming that the miR-92 binding sites are a critical regulatory region. Finally, we showed that miR-92 expression was upregulated by 17β-estradiol and downregulated by tamoxifen in MCF7 cells (ERα+ ERβ+) but not in ERβ negative cells (BT20 and MDAMB453), suggesting ERs can mediate miR-92 regulation. Our results demonstrate that ERβ1 expression in breast cancer is regulated by miRNA-92. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4139.