Endocrine disruptor regulation of microRNA expression in breast carcinoma cells

Abstract

Background: Several environmental agents termed "endocrine disrupting compounds" or EDCs have been reported to bind and activate the estrogen receptor-α (ER). The EDCs DDT and BPA are ubiquitously present in the environment, and DDT and BPA levels in human blood and adipose tissue are detectable in most if not all women and men. ER-mediated biological responses can be regulated at numerous levels, including expression of coding RNAs (mRNAs) and more recently non-coding RNAs (ncRNAs). Of the ncRNAs, microRNAs have emerged as a target of estrogen signaling. Given the important implications of EDC-regulated ER function, we sought to define the effects of BPA and DDT on microRNA regulation and expression levels in estrogen-responsive human breast cancer cells.

Methodology/principal findings: To investigate the cellular effects of DDT and BPA, we used the human MCF-7 breast cancer cell line, which is ER (+) and hormone sensitive. Our results show that DDT and BPA potentiate ER transcriptional activity, resulting in an increased expression of receptor target genes, including progesterone receptor, bcl-2, and trefoil factor 1. Interestingly, a differential increase in expression of Jun and Fas by BPA but not DDT or estrogen was observed. In addition to ER responsive mRNAs, we investigated the ability of DDT and BPA to alter the miRNA profiles in MCF-7 cells. While the EDCs and estrogen similarly altered the expression of multiple microRNAs in MCF-7 cells, including miR-21, differential patterns of microRNA expression were induced by DDT and BPA compared to estrogen.

Conclusions/significance: We have shown, for the first time, that BPA and DDT, two well known EDCs, alter the expression profiles of microRNA in MCF-7 breast cancer cells. A better understanding of the molecular mechanisms of these compounds could provide important insight into the role of EDCs in human disease, including breast cancer.

Figure 1. ERE-luciferase assay in MCF-7 cells.

Cells were incubated overnight in media containing 5% charcoal-stripped FBS and were then transfected with an ERE-luciferase plasmid. After 4 hrs, drugs were added to the cells as indicated and luciferase levels were measured 18 hrs. later.

Figure 2. Quantitative PCR confirmation of select genes altered by estrogen, BPA, and DDT in MCF-7 cells.

MCF-7 cells were treated with drug as indicated and total RNA was isolated, reverse-transcribed into cDNA, and subjected to real-time RT-PCR analysis for quantitation. Results are shown for (A) SERPINB5 (maspin) and (B) Bcl2 gene expression. Treatment of MCF-7 cells was as follows: Ethanol (vehicle), 1 nM E₂, 10 µM BPA, and 10 µM DDT. Results are expressed as the mean fold induction ± S.E.M. (***, p<0.001; **, p = 0.01 – 0.001; *, p = 0.05 - 0.01).

Figure 3. MicroRNA microarray heatmaps of MCF-7 cells treated with vehicle, estrogen, BPA, or DDT.

(A) Estrogen versus control (B) BPA versus control and (C) DDT versus control. Arrowheads represent expression of miR-21.

Figure 4. MicroRNA microarray of MCF-7 breast cancer cells treated with E₂, BPA and DDT.

Venn diagram showing the relative expression profiles of microRNA's from MCF-7 cells treated with estrogen vs BPA vs DDT.

Figure 5. Effects of E₂, BPA and DDT on miR-21 gene expression in MCF-7 and MCF-7F cells.

MCF-7 cells (A) and MCF-7F cells (B) were treated with either vehicle, 1 nM E₂, 10 µM BPA, or 10 µM DDT. Total microRNA was extracted followed by RT-PCR. * = p<0.05, ** = p<0.001.

Figure 6. The effect of BPA and DDT on endogenous miR-21 target genes in MCF-7 cells.

MCF-7 cells were treated for 18 hrs. with 0, 1, 10 and 25 µM BPA or DDT followed by RT-PCR of PDCD4 and maspin.