Xenoestrogen-induced epigenetic repression of microRNA-9-3 in breast epithelial cells

Abstract

Early exposure to xenoestrogens may predispose to breast cancer risk later in adult life. It is likely that long-lived, self-regenerating epithelial progenitor cells are more susceptible to these exposure injuries over time and transmit the injured memory through epigenetic mechanisms to their differentiated progeny. Here, we used progenitor-containing mammospheres as an in vitro exposure model to study this epigenetic effect. Expression profiling identified that, relative to control cells, 9.1% of microRNAs (82 of 898 loci) were altered in epithelial progeny derived from mammospheres exposed to a synthetic estrogen, diethylstilbestrol. Repressive chromatin marks, trimethyl Lys27 of histone H3 (H3K27me3) and dimethyl Lys9 of histone H3 (H3K9me2), were found at a down-regulated locus, miR-9-3, in epithelial cells preexposed to diethylstilbestrol. This was accompanied by recruitment of DNA methyltransferase 1 that caused an aberrant increase in DNA methylation of its promoter CpG island in mammosphere-derived epithelial cells on diethylstilbestrol preexposure. Functional analyses suggest that miR-9-3 plays a role in the p53-related apoptotic pathway. Epigenetic silencing of this gene, therefore, reduces this cellular function and promotes the proliferation of breast cancer cells. Promoter hypermethylation of this microRNA may be a hallmark for early breast cancer development, and restoration of its expression by epigenetic and microRNA-based therapies is another viable option for future treatment of this disease.

Figure 1

Preexposure of MDECs to diethylstilbestrol and immunofluorescence analysis of nuclear ER-α. A, treatment scheme. Breast progenitor cells were propagated as nonadherent spherical colonies, called mammospheres, and treated with 70 nmol/L diethylstilbestrol or DMSO solvent control for 3 wk. To induce differentiation, cells were seeded on a collagen substratum in the absence of diethylstilbestrol for 3 wk. Expression profiling (microRNA microarray), immunofluorescence image (IFA), and epigenetic [chromatin immunoprecipitation-PCR (ChIP-PCR) and MassARRAY] analyses were then done on the progeny epithelial cells. B, increased internalization of ER-α in diethylstilbestrol-preexposed MDECs. After the preexposure to diethylstilbestrol (DES) or DMSO, MDECs were subjected to immunofluorescence analysis. The percentage of subcellular localization of ER-α–positive cells, independently scored by two researchers, is shown in the bar chart. Columns, mean of five independent sets of MDEC samples; bars, SE. P < 0.001 (Student’s t test). C, nuclear trafficking of ER-α in MDECs on diethylstilbestrol treatment. DMSO-preexposed MDECs were exposed to 70 nmol/L diethylstilbestrol in the indicated time points. Translocation of ER-α protein (green) from the cytoplasm to the nucleus was observed, suggesting functional estrogen signaling. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The turquoise signals (merged) highlight the localization of ER-α in the nucleus.

Figure 2

microRNA expression profiling of diethylstilbestrol-preexposed MDECs. A, differentially expressed microRNA loci in diethylstilbestrol-preexposed MDECs. A total of 82 differentially expressed loci in control and preexposed cells are shown in the scatter plot (see Supplementary Table S1 for a complete list of the differentially expressed loci). The scatter plot represents the averaged values of three experiments. B and C, validation of differentially expressed loci by qRT-PCR. Gene-specific qRT-PCR on four independent sets of DMSO-and diethylstilbestrol-preexposed MDECs was done to validate four up-regulated loci (B) and six down-regulated loci (C). U6 was used as internal control for microRNA expression. Mean ± SE (n = 4). ***, P < 0.001; **, P < 0.01; *, P < 0.05 (Student’s t test).

Figure 3

Recruitment of epigenetic markers at the miR-9-3 locus. DNA from DMSO-or diethylstilbestrol-preexposed MDECs was immunoprecipitated with antibodies specific for H3K27me3, H3K9me2, and DNA methyltransferase 1. Immunoprecipitated DNA was subjected to quantitative PCR to measure enrichment at regions from 3.5 kb upstream to 1 kb downstream of the miR-9-3 TSS. Mean ± SE (n = 6) of two independent experiments. Bottom, a diagram of regions surveyed by real-time PCR in miR-9-3.

Figure 4

DNA methylation analysis of miR-9-3 locus. A, quantitative methylation analysis of miR-9-3 CpG sites by MassARRAY. A total of 39 CG dinucleotides, located approximately −0.1 to 0.3 kb of the miR-9-3 transcriptional start site (TSS), were interrogated in four sets of diethylstilbestrol- and DMSO-preexposed MDECs and seven breast cancer cell lines. CpG unit measurements in the analyzed region of miR-9-3 for all samples are depicted in the heat map. Color intensity represents the methylation ratios (see scale bar). Spots represent methylation data from each CpG unit. B, expression profile of miR-9-3 in breast cancer cell lines. qRT-PCR was conducted to examine miR-9-3 expression in three ER-α–positive (T47D, MCF-7, and MDA-MB-134) and four ER-α–negative (MCF-10A, SKBR3, MDA-MB-435S, and MDA-MB-231) breast cancer cell lines. Mean ± SE (n = 3). ***, P < 0.001 (Student’s t test), compared with normal MDECs. C, promoter hypermethylation of miR-9-3 in breast tumors. Quantitative methylation of miR-9-3 CpG sites on breast primary tissues was analyzed by MassARRAY. Five CpG units upstream of the miR-9-3 TSS were interrogated in 115 clinical specimens, including 102 tumors and 13 normal breast tissues. D, correlation between miR-9-3 methylation and ER-α status in patient tumors was assessed by the Mann Whitney rank-sum test (P = 0.001).

Figure 5

Epigenetic silencing of miR-9-3 mediated by ER-α–dependent and ER-α–independent pathways. A and B, effects of epigenetic inhibitors on miR-9-3 expression of breast cancer cell lines. Four ER-α–positive (A) and six ER-α–negative (B) breast cancer cell lines were pretreated with 5-aza-2′-deoxycytidine (DAC; 1 µmol/L) and/or trichostatin A (TSA; 1 µmol/L) and then stimulated with diethylstilbestrol for 6 h. Total RNA was collected for qRT-PCR analysis to monitor miR-9-3 expression. U6 was used as internal control. Mean ± SE (n = 3). ***, P < 0.001; **, P < 0.01; *, P < 0.05 (Student’s t test), compared with DMSO-treated cells. C, restoration of miR-9-3 expression by the ER-α antagonist, ICI182780. An ER-α–positive breast cancer cell line, MCF-7, was treated with 5-aza-2′-deoxycytidine (1 µmol/L), trichostatin A (1 µmol/L), and/or ER-α antagonist, ICI182780 (1 µmol/L), before 6 h diethylstilbestrol stimulation. Total RNA was addressed to qRT-PCR analysis of miR-9-3. U6 was the internal control. Mean ± SE (n = 3). ***, P < 0.001; **, P < 0.01; *, P < 0.05 (Student’s t test).

Figure 6

Direct and indirect target genes of miR-9-3. A, microarray profiles of miR-9-3 targets. MCF-7cells were transfected with two forms (miR-9 or miR-9* mimics) of miR-9-3. After 48 h, total RNA was collected for microarray analysis. Common genes derived microarray profiling and TargetScan database were shown in the heat map (see also Supplementary Table S2 for the list of target genes). The indicated genes are found in signaling network of p53. B, signaling network of p53 regulated by miR-9-3. Network interaction of the p53 pathway was constructed using the Ingenuity Pathway program, which identified 34 significant genes as the miR-9-3 targets. Genes in red and green ovals are up-regulated and down-regulated genes, respectively. Genes in blue open rectangles are correlated to the p53 pathway. C, confirmation of potential miR-9-3 targets. The expression level of selected p53 pathway-related genes was examined by qRT-PCR in miR-9 transfectants (top) and diethylstilbestrol-preexposed MDECs (bottom). β-Actin was used as internal control. D, induction of apoptosis by miR-9-3. microRNA mimics (miR-9 and miR-9*) were separately transfected into MCF-7cells for 48 h. Apoptosis assay was done, as described in Materials and Methods, on days 4 and 7 after the transfection. Scrambled oligonucleotides were used as negative controls. Mean ± SE (n = 3). ***, P < 0.001; **, P < 0.01; *, P < 0.05 (Student’s t test), compared with control cells.