title: miR-200 promotes the mesenchymal to epithelial transition by suppressing multiple members of the Zeb2 and Snail1 transcriptional repressor complexes.
abstract: The miR-200 family promotes the epithelial state by suppressing the Zeb1/Zeb2 epithelial gene transcriptional repressors. To identify other miR-200-regulated genes, we isolated mRNAs bound to transfected biotinylated miR-200c in mouse breast cancer cells. In all, 520 mRNAs were significantly enriched in miR-200c binding at least twofold. Putative miR-200-regulated genes included Zeb2, enriched 3.5-fold in the pull down. However, Zeb2 knockdown does not fully recapitulate miR-200c overexpression, suggesting that regulating other miR-200 targets contributes to miR-200's enhancement of epithelial gene expression. Candidate genes were highly enriched for miR-200c seed pairing in their 3'UTR and coding sequence and for genes that were downregulated by miR-200c overexpression. Epidermal growth factor receptor and downstream MAPK signaling pathways were the most enriched pathways. Genes whose products mediate transforming growth factor (TGF)-β signaling were also significantly overrepresented, and miR-200 counteracted the suppressive effects of TGF-β and bone morphogenic protein 2 (BMP-2) on epithelial gene expression. miR-200c regulated the 3'UTRs of 12 of 14 putative miR-200c-binding mRNAs tested. The extent of mRNA binding to miR-200c strongly correlated with gene suppression. Twelve targets of miR-200c (Crtap, Fhod1, Smad2, Map3k1, Tob1, Ywhag/14-3-3γ, Ywhab/14-3-3β, Smad5, Zfp36, Xbp1, Mapk12, Snail1) were experimentally validated by identifying their 3'UTR miR-200 recognition elements. Smad2 and Smad5 form a complex with Zeb2 and Ywhab/14-3-3β and Ywhag/14-3-3γ form a complex with Snail1. These complexes that repress transcription assemble on epithelial gene promoters. miR-200 overexpression induced RNA polymerase II localization and reduced Zeb2 and Snail1 binding to epithelial gene promoters. Expression of miR-200-resistant Smad5 modestly, but significantly, reduced epithelial gene induction by miR-200. miR-200 expression and Zeb2 knockdown are known to inhibit cell invasion in in vitro assays. Knockdown of each of three novel miR-200 target genes identified here, Smad5, Ywhag and Crtap, also profoundly suppressed cell invasion. Thus, miR-200 suppresses TGF-β/BMP signaling, promotes epithelial gene expression and suppresses cell invasion by regulating a network of genes.
authors: Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA, USA.|Animal Cell Technology Unit, Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Oeiras, Portugal.|Instituto de Biologia Experimental e Tecnológica (IBET), Oeiras, Portugal.
authors: R
authors: R
authors: Perdigão-Henriques
authors: Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA, USA.
authors: F
authors: F
authors: Petrocca
authors: Department of Biostatistics, Harvard School of Public Health, Boston, MA, USA.
authors: G
authors: G
authors: Altschuler
authors: Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA, USA.
authors: M P
authors: MP
authors: Thomas
authors: Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA, USA.
authors: M T N
authors: MT
authors: Le
authors: Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA, USA.
authors: S M
authors: SM
authors: Tan
authors: Department of Biostatistics, Harvard School of Public Health, Boston, MA, USA.|Sheffield Institute for Translational Neuroscience, Department of Neuroscience, University of Sheffield, Sheffield, UK.
authors: W
authors: http://orcid.org/0000-0002-8621-3271
authors: W
authors: Hide
authors: Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA, USA.|Department of Pediatrics, Harvard Medical School, Boston, MA, USA.
authors: J
authors: J
authors: Lieberman
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects / Q000235:genetics*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D053263:Gene Regulatory Networks; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D041681:NIH 3T3 Cells; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology; D000074165:Zinc Finger E-box Binding Homeobox 2
chemical_list: D020413:3' Untranslated Regions; D055396:Bone Morphogenetic Protein 2; D015820:Cadherins; D018398:Homeodomain Proteins; C533896:MIRN200 microRNA, human; D035683:MicroRNAs; C543719:Mirn200 microRNA, mouse; D012097:Repressor Proteins; C000604407:SNAI1 protein, human; C000604429:Snai1 protein, mouse; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; C120602:ZEB2 protein, mouse; D000074165:Zinc Finger E-box Binding Homeobox 2