The Micro-Ribonucleic Acid (miRNA) miR-206 Targets the Human Estrogen Receptor-α (ERα) and Represses ERα Messenger RNA and Protein Expression in Breast Cancer Cell Lines

Abstract

Micro-RNAs are small noncoding RNAs, which diminish the stability and/or translation of mRNAs. This study examined whether miR-206, previously shown to be elevated in estrogen receptor (ER)α-negative breast cancer, regulates the expression of ERα. Two putative miR-206 sites, (hERα1 and hERα2), were found in silico within the 3′-untranslated region of human ERα mRNA. Transfection of MCF-7 cells with pre-miR-206 or 2′-O-methyl antagomiR-206 specifically decreased or increased, respectively, ERα mRNA levels. Overexpression of pre-miR-206 reduced ERα and β-actin protein levels, with no effect on ERβ, E-cadherin, or glyceraldehyde-3-phosphate dehydrogenase. Reporter constructs containing the hERα1 or hERα2 binding sites inserted into the 3′-untranslated region of the luciferase mRNA conferred a 1.6- and 2.2-fold repression of luciferase activity, respectively, in HeLa cells. Both miR-206 sites responded accordingly to exogenous hsa-pre-miR-206 and 2′-O-methyl antagomiR-206, and both sites were rendered inactive by mutations that disrupted hybridization to the 5′-seed of miR-206. A C→T single nucleotide polymorphism in the hERα1 site increased repression of luciferase activity to approximately 3.3-fold in HeLa cells. MiR-206 levels were higher in ERα-negative MB-MDA-231 cells than ERα-positive MCF-7 cells, but only the ERα1 site mediated significantly more repression in reporter constructs. MiR-206 expression was strongly inhibited by ERα agonists, but not by an ERβ agonist or progesterone, indicating a mutually inhibitory feedback loop. These findings provide the first evidence for the posttranscriptional regulation of ERα by a micro-RNA in the context of breast cancer.