Novel estrogen-responsive genes (ERGs) for the evaluation of estrogenic activity

Estrogen action is mediated by various genes including estrogen-responsive genes (ERGs). ERGs have been used as markers for gene expression and as reporter-genes, while gene expression profiling using a set of ERGs has been used as statistically reliable transcriptomic assays, such as DNA microarray assays and RNA-seq. However, the quality of ERGs has not been extensively examined. Here, we found a set of 300 ERGs newly identified by six sets of RNA-seq data obtained from estrogen-treated and control human breast cancer MCF-7 cells. The ERGs exhibited statistical stability as judged by the coefficient of variation (CV) analysis, and their usefulness as markers for estrogenic activity by examining the stability of data in the study of correlation analysis, and functional association with estrogen action through database searches. A set of the top 30 genes based on CV ranking were further evaluated quantitatively by RT-PCR and qualitatively by a functional analysis using GO and KEGG databases and by a mechanistic analysis to classify ERa/ß-dependent or ER-independent types of transcriptional regulation. The 30 ERGs were characterized by (1) the enzymes, such as metabolic enzymes, proteases and protein kinases, (2) the genes with specific cell functions, such as cell-signaling mediators, tumor-suppressors and the roles in breast cancer, (3) the association with transcriptional regulation, and/or (4) estrogen-responsiveness. Therefore, the ERGs identified here could represent various cell functions and cell signaling pathways including estrogen signaling, and thus, would be useful to evaluate estrogenic activity. Overall design: MCF-7 cells at a density of 1.0×10^6 cells per well were cultured in RPMI1640 supplemented with 10% DCC-FBS for 3 days under the conditions of 37°C in 5% CO2. The cells were treated in RPMI1640 including 10 nM E2, 1 µM ICI or 0.1% DMSO (vehicle) for 2 more days.