Modification of gene expression induced by siRNA targeting of estrogen receptor a in MCF7 human breast cancer cells

To establish a model of endocrine resistant breast cancer that is associated with loss of estrogen receptor (ER), MCF7 cells were transfected with several plasmid constructs intended to produce intracellular double stranded hairpin RNA to be processed into siRNA directed against different regions of the ER alpha mRNA. Stably transformed cells were propagated in long-term culture. One of these lines, designated pit, was selected for further analysis. pit cells exhibited reduced levels of ERa mRNA and protein as well as several estrogen-regulated genes assessed by real-time PCR and were unresponsive to addition of estradiol and tamoxifen. compared with Higher levels of ER beta were measurable as cot parental MCF7 cells. There was an unexpected decrease in expression in members of the EGFR family in contrast with e other observations reported for ER-negative tumours or some established endocrine-independent lines. Microarray gene analysis comparing expression in parental MCF7 with pit cells in both serum-synchronised and non-synchronised conditions highlighted a spectrum of other genes that were expressed at different levels compared to the parental MCF7 cells. Genes showing the greatest change were mostly common between synchronized and Unsynchronised cells; GRB7, PSMD7, KRT19, KRT18, AKT1, SYNCRIP, CYB5A and EVL for down-regulated in p11 and QDPR, VIM, CD68, CA9, STMN1, CDK2, CTSC for Up-regulated in pit cells. Notably, the decreased expression of epithelial keratins 18 acrophage and 19 and an increase in vimentin and in a m, marker CD68, is suggestive of an epithelial to mesothelial Further characterisation of these cells particularly transition, Fut with respect to the factors controlling their growth may contribute to a better understanding of the behaviour of cells that have become endocrine independent by loss of ER function.