Fig. 2
miRNA-1199-5p inhibits an EMT and tumour cell migration and invasion. a miR-1199-5p transcript and miR-1199 promoter activity levels decrease during a TGFβ-induced EMT. Green line: expression profile of miR-1199-5p during EMT in NMuMG/E9 cells as determined by RNA sequencing. Grey bars: miR-1199 promoter activity during an EMT. NMuMG/E9 cells were treated with TGFβ for the time points indicated and transfected with a Renilla luciferase reporter along with either a miR-1199 promoter Firefly luciferase reporter (pGL4 miR-1199 promoter) or a control reporter (pGL4; Supplementary Fig. 3b). Relative luminescence (Firefly/Renilla) was calculated and normalized to the control reporter (mean fold changes ± s.e.m.; n = 3; significance determined by an unpaired, two-sided t test; *P < 0.05, **P < 0.01). b, c NMuMG/E9 cells were transiently transfected with the miRNA mimics indicated and cultured in presence of TGFβ for 4 days (4-day TGFβ). Bright-field images of NMuMG/E9 cells illustrate the differences in cell morphology. Immunofluorescence images of NMuMG/E9 cells visualize different epithelial and mesenchymal cell structures as indicated. Scale bars: 100 μm. d, e Immunoblot (d) and quantitative RT-PCR mRNA (e) expression analysis of NMuMG/E9 cells transiently transfected with miRNA mimics for the EMT markers indicated (mean fold changes ± s.e.m.; n = 6; significance determined by an unpaired, two-sided t test, Student’s t test; **P < 0.01, ***P < 0.001, ****P < 0.0001). f, g Mesenchymal (>20 days TGFβ) Py2T (f) and 4T1 (g) cells were transiently transfected with the miRNA mimics indicated and re-plated within a FCS gradient of a Boyden chamber migration or invasion insert. Nuclei of transmigrated and invaded cells were quantified after 18 h (mean fold changes ± s.e.m.; migration: n = 3; invasion Py2T: n = 3, 4T1: n = 4; significance determined by an unpaired, two-sided t test; *P < 0.05, **P < 0.01, ****P < 0.0001)
