Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines.

Predicting the impact of microRNAs (miRNAs) on target proteins is challenging because of their different regulatory effects at the transcriptional and translational levels. In this study, we applied a novel protein lysate microarray (LMA) technology to systematically monitor for target protein levels after high-throughput transfections of 319 pre-miRs into breast cancer cells. We identified 21 miRNAs that downregulated the estrogen receptor-alpha (ERalpha), as validated by western blotting and quantitative real time-PCR, and by demonstrating the inhibition of estrogen-stimulated cell growth. Five potent ERalpha-regulating miRNAs, miR-18a, miR-18b, miR-193b, miR-206 and miR-302c, were confirmed to directly target ERalpha in 3'-untranslated region reporter assays. The gene expression signature that they repressed highly overlapped with that of a small interfering RNA against ERalpha, and across all the signatures tested, was most closely associated with the repression of known estrogen-induced genes. Furthermore, miR-18a and miR-18b showed higher levels of expression in ERalpha-negative as compared with ERalpha-positive clinical tumors. In summary, we present systematic and direct functional evidence of miRNAs inhibiting ERalpha signaling in breast cancer, and demonstrate the high-throughput LMA technology as a novel, powerful technique in determining the relative impact of various miRNAs on key target proteins and associated cellular processes and pathways.