title: Identification of miR-193b targets in breast cancer cells and systems biological analysis of their functional impact.
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.
authors.affiliation: VTT Technical Research Centre of Finland, Medical Biotechnology, P.O. Box 106, FI-20521, Turku, Finland. suvi-katri.leivonen@vtt.fi
authors.forename: Suvi-Katri
authors.initials: SK
authors.lastname: Leivonen
authors.forename: Anne
authors.initials: A
authors.lastname: Rokka
authors.forename: Päivi
authors.initials: P
authors.lastname: Ostling
authors.forename: Pekka
authors.initials: P
authors.lastname: Kohonen
authors.forename: Garry L
authors.initials: GL
authors.lastname: Corthals
authors.forename: Olli
authors.initials: O
authors.lastname: Kallioniemi
authors.forename: Merja
authors.initials: M
authors.lastname: Perälä
mesh_terms: D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D052138:Genes, Neoplasm; D017930:Genes, Reporter; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006836:Hydro-Lyases; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D049490:Systems Biology; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
chemical_list: D012696:Glycine Hydroxymethyltransferase; D006836:Hydro-Lyases; D006913:Hydroxysteroid Dehydrogenases; D049410:Luciferases, Renilla; D008565:Membrane Proteins; D035683:MicroRNAs; D017027:Protein Tyrosine Phosphatases; D020543:Proteome; D014446:Tyrosine 3-Monooxygenase; C546818:MIRN193 microRNA, human; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); C484872:HACD2 protein, human