Abstract
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-α (ERα) 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 14-3-3ζ (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 ERα, 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.
Highlights
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
Thereafter, the cDNAs were diluted 1/10 and the Taqman quantitative real-time PCR analysis performed with Applied Biosystems 7900HT instrument using specific primers and probes for YWHAZ, SHMT2, AKR1C2, PTPLB, MCM7, HSP90AB1, and glyseraldehyde 3-phosphate-dehydrogenase (GAPDH) designed by the Universal Probe Library Assay Design Center (Roche Applied Biosciences, Basel, Switzerland)
Identification of miR-193b Targets by iTRAQ Proteomics—We have previously shown, that miR-193b directly targets ER␣, induces cell cycle arrest, and inhibits growth when overexpressed in breast cancer cells [7]
Summary
Cell Culture and Transfections—MCF-7 cells were obtained from Interlab Cell Line Collection (ICLC, Italy), and cultured in Dulbecco’s modified Eagles medium (DMEM) (1 g/l glucose) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin/ streptomycin. ITRAQ Labeling—MCF-7 cells were transfected with 20 nM pre-miR negative control and pre-miR-193b (Ambion) in six-well plates. Protein digestion and labeling were done according to iTRAQ 4-plex Reagent Kit (Applied Biosystems, Foster City, CA). Each sample was digested by 10 g of sequence grade modified trypsin (Promega) at ϩ37 °C overnight, and labeled with the iTRAQ reagents. Protein identification and quantitation were carried out using the ProteinPilot software (version 2.0.1, Applied Biosystems, MDS-Sciex) with the ParagonTM search algorithm [9]. Thereafter, the cDNAs were diluted 1/10 and the Taqman quantitative real-time PCR (qRT-PCR) analysis performed with Applied Biosystems 7900HT instrument using specific primers and probes for YWHAZ, SHMT2, AKR1C2, PTPLB, MCM7, HSP90AB1, and glyseraldehyde 3-phosphate-dehydrogenase (GAPDH) designed by the Universal Probe Library Assay Design Center (Roche Applied Biosciences, Basel, Switzerland) (supplementary Table S1). Enrichment of the predictions was calculated by comparing the proportion of predicted targets among the miR-193b down-regulated proteins to predicted targets among all proteins detected in the control sample, or by comparing the proportion of predicted targets among the miR-193b down-regulated transcripts to predicted targets among all genes on the array
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