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.