Fig 2: miR-1199-5p directly controls Zeb1 expression. a Species-conserved miR-1199-5p-binding site in the 3′ UTR of Zeb1. The scheme represents: top: sequence alignments of predicted binding sites of miR-1199-5p (red; mouse: position 239–246) in Zeb1 3′ UTRs of different species. Middle: alignment of the 8mer seed match in mouse miR-1199-5p. Bottom: mutated miR-1199-5p seed sequence in the 3′ UTR of Zeb1 mRNA utilized for the control reporter construct (Zeb1 3′ UTR mut) in d. Exchanged nucleotides are underlined. b, c Forced expression of miR-1199-5p reduces Zeb1 nuclear localization and protein levels during an EMT. NMuMG/E9 cells were transiently transfected with miRNA mimics as indicated and cultured in the absence (0 day) or presence of TGFβ (4 days). Immunofluorescence (b) and immunoblotting (c) analyses illustrate the differences in Zeb1 protein levels. Scale bar: 100 μm. d Post-transcriptional regulation of Zeb1 by miR-1199-5p. NMuMG/E9 cells were transfected with the miRNA mimics indicated, with a Renilla luciferase reporter construct and with either a Zeb1 3′ UTR wild-type (wt) or a Zeb1 3′ UTR mutant (mut) Firefly luciferase reporter construct. Relative luminescence (Firefly/Renilla) was calculated and normalized to miR-Ctr-transfected cells (mean fold changes±s.e.m.; n = 3; significance determined by an unpaired, two-sided t test; *P < 0.05)

Fig 3: Mesenchymal cancer cell populations isolated from co-cultures of epithelial prostate cancer cells and human macrophages.(A) Bright-phase microscopy: morphology changes in the mesenchymal cells PC3-EMT1, PC3-EMT12 and PC3-EMT14 as compared to the epithelial PC3-Epi prostate cancer cells. Scale bars are 200 µm.(B) Immunoblot: Expression of EMT markers in mesenchymal cancer cell lines PC3-EMT1, -EMT2, -EMT12, -EMT14 and -EMT17 compared to epithelial PC3-Epi.(C) Microarray: Gene expression analyses comparing mesenchymal to epithelial cancer cell lines. Venn diagram-I (VD-I) depicts a common EMT-associated signature expressed in the mesenchymal cancer lines PC3-EMT1, -EMT12 and -EMT14 compared to epithelial PC3-Epi. VD-II – Gene signature from VD-I intersected with the signature of ZEB1 silenced cells PC3-EMT1 and -EMT14 using the shRNA-sh4, relative to scramble (Scr) control shRNA.(D) Bright-phase microscopy: PC3-EMT14 cells transfected with ZEB1-shRNAs: sh2, sh4, or Scr. Scale bars are 100 µm.(E) Immunoblot: Expression of EMT markers in PC3-EMT1 and -EMT14 transfected with ZEB1-shRNAs compared to Scr or non-transfected controls.(F) Heat map: 50 genes signature identified in VD-II (panel C). Upregulated genes are in red, downregulated in blue. Fold changes in mesenchymal cancer cells are relative to epithelial PC3-Epi, and in sh4-transfected cells are relative to the Scr control.The immunoblots shown are representative of two independent experiments with similar results. See also Figure S1 and Table S1.

Fig 4: Silencing of STAT3 down-regulated the expression of integrin α3 and knockdown of integrin α3 inhibits metastatic phenotypes of colon cancer cells. (A) Expression of ZEB1, integrin α3, and GOLPH3 of control and STAT3 siRNA transfected cells were assessed using western blotting and qRT-PCR. (B) Integrin α3 expression in control and integrin α3 siRNA transfected cells were assessed using western blotting and qRT-PCR. (C,D) The effect of integrin α3 knockdown in HCT116 and HCT8 cells migration or invasion were examined by transwell assay. The number of migratory or invasive cells were counted and normalized to the NC group in five randomly selected fields. Representative micrographs are also shown (×200 magnification). (E) Working model. GOLPH3 interacts with p-STAT3 and regulates ZEB1 and integrin α3 to promote the metastasis of CRC. Scale bar, 100 µm. For (A,B), results were reproduced in three independent experiments, and representative immunoblots are shown. **p < 0.01, ***p < 0.001.

Fig 5: ET-1/ETAR axis downregulates miR-200b/c via ZEB1.a Expression of ZEB1 and ETAR proteins in HEY cells transfected for 48 h with siRNA control (si-Ctr) or si-ETAR or treated with MAC and stimulated or not for 48 h with ET-1 analyzed by WB. β-actin is used as loading control. b Luciferase activity in HEY cells co-transfected for 48 h with si-Ctr, si-ETAR, and the ZEB1 promoter reporter plasmid and stimulated with ET-1 and/or MAC for 24 h.Values are the mean ± SD expressed as fold induction (n = 3; *p < 0.0001 vs. si-Ctr; **p < 0.0001 vs. ET-1 stimulated si-Ctr). c qRT-PCR for miR-200b/c expression in HEY cells transfected with si-Ctr or si-ZEB1 and stimulated with ET-1 for 72 h in the absence or in the presence of MAC. U6 is used to normalize. Values are the mean ± SD (n = 3; *p < 0.001 vs. unstimulated Ctr; **p < 0.001 vs. ET-1-stimulated Ctr). d Pri-miR-200b/c expression in cells transfected and treated as in c for 48 h is evaluated by qRT-PCR and normalized to cyclophilin-A. Values are the mean ± SD (n = 3; *, p < 0.01 vs. unstimulated Ctr; **p < 0.001 vs. ET-1-stimulated Ctr). e Schematic representation of the ZEB1 binding sites contained in the miR-200b/c promoter reporter plasmids. f Luciferase activity in HEY cells co-transfected for 48 h with si-Ctr, si-ETAR, or si-ZEB1 and the reporter plasmids for miR-200b/c promoters and stimulated or not with ET-1 and/or MAC for 24 h. Values are the mean ± SD expressed as fold induction (n = 3; *p < 0.001 vs. unstimulated Ctr; **p < 0.001 vs. ET-1-stimulated Ctr).