Effects of oestrogen on microRNA expression in hormone-responsive breast cancer cells

Abstract

Oestrogen receptor alpha (ERα) is a ligand-dependent transcription factor that mediates oestrogen effects in hormone-responsive cells. Following oestrogenic activation, ERα directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) represent a class of small noncoding RNAs that function as negative regulators of protein-coding gene expression. They are found aberrantly expressed or mutated in cancer, suggesting their crucial role as either oncogenes or tumour suppressor genes. Here, we analysed changes in miRNA expression in response to oestrogen in hormone-responsive breast cancer MCF-7 and ZR-75.1 cells by microarray-mediated expression profiling. This led to the identification of 172 miRNAs up- or down-regulated by ERα in response to 17β-oestradiol, of which 52 are similarly regulated by the hormone in the two cell models investigated. To identify mechanisms by which ERα exerts its effects on oestrogen-responsive miRNA genes, the oestrogen-dependent miRNA expression profiles were integrated with global in vivo ERα binding site mapping in the genome by ChIP-Seq. In addition, data from miRNA and messenger RNA (mRNA) expression profiles obtained under identical experimental conditions were compared to identify relevant miRNA target transcripts. Results show that miRNAs modulated by ERα represent a novel genomic pathway to impact oestrogen-dependent processes that affect hormone-responsive breast cancer cell behaviour. MiRNome analysis in tumour tissues from breast cancer patients confirmed a strong association between expression of these small RNAs and clinical outcome of the disease, although this appears to involve only marginally the oestrogen-regulated miRNAs identified in this study.

Fig. 1

Characterization of ERα-positive breast cancer cell lines. a Expression level of ERα by Western blot analysis of protein extracts from MCF-7 (lane 1), ZR-75.1 (lane 2) and SKBR3 (lane 3) cells. b Monitoring of cyclin gene expression in G1-synchronised MCF-7 and ZR-75.1 cell lines following stimulation with a mitogenic dose of E2

Fig. 2

Effects of ERα on miRNome of human breast cancer cell lines. Time-course analysis of miRNA expression profiles in MCF-7 and ZR-75.1 cell lines after cells exposure to E2 for the indicated times. Oestrogen-regulated miRNAs are grouped as follows: regulated in MCF-7 cells only (a), in both cell lines (b) or in ZR-75.1 cells only (c). Data displayed represent the ratio between the fluorescence intensity values of each miRNA at the indicated time after exposure to 10⁻⁸ M E2 vs the corresponding 0 h time point. MicroRNAs marked in red represent those regulated in BC cell lines in vitro and those displaying differential expression between primary breast tumour subgroups (see Fig. 4)

Fig. 3

Graphic representation of timed fold-change variations of selected E2-regulated miRNAs in MCF-7 and ZR-75.1 cell lines. MicroRNAs shown are members of clusters (a, c) or are encoded as independent transcript (b, d)

Fig. 4

Analysis of miRNA expression in 30 breast cancer samples. a Hierarchical clustering of 161 miRNAs oestrogen-regulated in vitro in at least one cell line. The blue bars denote ER+ samples. Numbers at the top indicate individual tumour number (see Supplemental Table S3 for details). The two main branches were used in survival analysis: b Kaplan–Meier survival analysis and log rank test concerning all samples [30] (p < 0.05 for DFS, top; p < 0.08 for OS, bottom) or c limited to patients receiving adjuvant Tamoxifen (p < 0.09 for DFS, top; NS for OS, bottom). ‘N’ on the curves denotes the censored events in each group. The miRNAs marked in red are in common between the miRNAs classifying breast tumours and those E2-regulated in BC cell lines