Anti-SHMT2 monoclonal antibody, clone 6F8

Identification of protein targets for microRNAs (miRNAs) is a significant challenge due to the complexity of miRNA-mediated regulation. We have previously demonstrated that miR-193b targets estrogen receptor-alpha (ER alpha) and inhibits estrogen-induced growth of breast cancer cells. Here, we applied a high-throughput strategy using quantitative iTRAQ (isobaric tag for relative and absolute quantitation) reagents to identify other target proteins regulated by miR-193b in breast cancer cells. iTRAQ analysis of pre-miR-193b transfected MCF-7 cells resulted in identification of 743 unique proteins, of which 39 were down-regulated and 44 up-regulated as compared with negative control transfected cells. Computationally predicted targets of miR-193b were highly enriched (sevenfold) among the proteins whose level of expression decreased after miR-193b transfection. Only a minority of these (13%) showed similar effect at the mRNA level illustrating the importance of post-transcriptional regulation. The most significantly repressed proteins were selected for validation experiments. These data confirmed 14-3-3 zeta (YWHAZ), serine hydroxyl transferase (SHMT2), and aldo-keto reductase family 1, member C2 (AKR1C2) as direct, previously uncharacterized, targets of miR-193b. Functional RNAi assays demonstrated that specific combinations of knockdowns of these target genes by siRNAs inhibited growth of MCF-7 cells, mimicking the effects of the miR-193b overexpression. Interestingly, the data imply that besides targeting ER alpha, the miR-193b effects include suppression of the local production of estrogens and other steroid hormones mediated by the AKR1C2 gene, thus provoking two separate molecular mechanisms inhibiting steroid-dependent growth of breast cancer cells. In conclusion, we present here a proteomic screen to identify targets of miR-193b, and a systems biological approach to mimic its effects at the level of cellular phenotypes. This led to the identification of multiple genes whose combinatorial knock-down likely mediates the strong anti-cancer effects observed for miR-193b in breast cancer cells. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.005322, 1-9, 2011.