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)alpha-negative breast cancer, regulates the expression of ERalpha. Two putative miR-206 sites, (hERalpha1 and hERalpha2), were found in silico within the 3'-untranslated region of human ERalpha mRNA. Transfection of MCF-7 cells with pre-miR-206 or 2'-O-methyl antagomiR-206 specifically decreased or increased, respectively, ERalpha mRNA levels. Overexpression of pre-miR-206 reduced ERalpha and beta-actin protein levels, with no effect on ERbeta, E-cadherin, or glyceraldehyde-3-phosphate dehydrogenase. Reporter constructs containing the hERalpha1 or hERalpha2 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 hERalpha1 site increased repression of luciferase activity to approximately 3.3-fold in HeLa cells. MiR-206 levels were higher in ERalpha-negative MB-MDA-231 cells than ERalpha-positive MCF-7 cells, but only the ERalpha1 site mediated significantly more repression in reporter constructs. MiR-206 expression was strongly inhibited by ERalpha agonists, but not by an ERbeta agonist or progesterone, indicating a mutually inhibitory feedback loop. These findings provide the first evidence for the posttranscriptional regulation of ERalpha by a micro-RNA in the context of breast cancer.

Widespread Estrogen-Dependent Repression of microRNAs Involved in Breast Tumor Cell Growth

“…Recently, two miRNAs (miR-206 and miR-21) were identified as being downregulated by E 2 in MCF-7 (24,25). In our miRNA microarray analysis, we indeed found an E 2 -dependent repression of miR-21 expression (Supplementary Table S1; also see Table 1 and Fig.…”

“…E 2 also regulates miRNA expression in the adult zebrafish (23). Finally, two miRNAs (miR-206 and miR-21) have recently been identified as being downregulated by E 2 in the MCF-7 breast tumor cell line (24,25). However, a high throughput analysis of E 2 -regulated miRNAs in breast tumor cell lines is lacking and the role of miRNAs in estradiol-dependent functions is poorly documented.…”

Caveolin proteins and estrogen signaling in the brain

“…Mutations of CAV1 are found in ~35% of ERα-positive human breast cancer samples [78], and CAV1 RNA and protein levels are reduced in many cases of human primary breast carcinomas [79]. Mechanistically, it has been theorized that these alterations in CAV1 function or expression contribute to the increased sensitivity of mammary tissue to estrogen by leading to an increase of ERα expression [78,80]. Moreover, overexpression of CAV1 in mammary tissue protects against the tumorigenic phenotype that mammary tissue lacking CAV1 shows [81].…”

MicroRNAs from biology to future pharmacotherapy: Regulation of cytochrome P450s and nuclear receptors

“…ERα regulates the expression of human CYP1B1, which catalyzes the metabolism of estradiol to a carcinogenic metabolite, 4-hydroxyestradiol (Tsuchiya et al, 2004). It has been demonstrated that human ERα is regulated by miR-206, whereas the activation of ERα results in the decreased expression of miR-206, showing mutually inhibitory regulation (Adams et al, 2007). After this report, it was reported that miR-221, miR-222 , and miR-22 (Xiong et al, 2010) also regulate human ERα expression.…”