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

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