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

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) -negative breast cancer, regulates the expression of ER . Two putative miR-206 sites, (hER 1 and hER 2), were found in silico within the 3 -untranslated region of human ER mRNA. Transfection of MCF-7 cells with pre-miR-206 or 2 -O-methyl antagomiR-206 specifically decreased or increased, respectively, ER mRNA levels. Overexpression of pre-miR-206 reduced ER and -actin protein levels, with no effect on ER , E-cadherin, or glyceraldehyde-3-phosphate dehydrogenase. Reporter constructs containing the hER 1 or hER 2 binding sites inserted into the 3 untranslated region of the luciferase mRNA conferred a 1.6and 2.2-fold repression of luciferase activity, respectively, in HeLa cells. Both miR-206 sites responded accordingly to exogenous hsa-premiR-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 C3T single nucleotide polymorphism in the hER 1 site increased repression of luciferase activity to approximately 3.3-fold in HeLa cells. MiR-206 levels were higher in ER -negative MB-MDA-231 cells than ER -positive MCF-7 cells, but only the ER 1 site mediated significantly more repression in reporter constructs. MiR-206 expression was strongly inhibited by ER agonists, but not by an ER agonist or progesterone, indicating a mutually inhibitory feedback loop. These findings provide the first evidence for the posttranscriptional regulation of ER by a micro-RNA in the context of breast cancer. (Molecular Endocrinology 21: 1132–1147, 2007)