Figure 3: miR-30c regulates EMT and directly targets TWF1.

(a) Genes suppressed by miR-30c in breast cancer cells. Top panel: left circle shows the microarray data of 293 genes significantly inhibited by miR-30c in MDA-MB-231 cells (see Supplementary Table S2). The right circle indicates the genes predicted as direct targets of miR-30c by Geneset2miR (by ≥4 of 11 algorithms) and overlapped genes are 77 (Supplementary Table S3). Bottom left panel: heatmap for eight anti-apoptotic genes inhibited by miR-30c. Bottom right panel: heatmap for predicted direct targets, downregulated in miR-30c-transfected cells. Green boxes are colour-coded based on log2-transformed expression reduction levels for four pairs of 30c/scrambled microarray comparisons. (b) Expression levels of TWF1 (measured by real-time PCR) upon transfection of miR-30c or the anti-miR-30c inhibitors into MDA-MB-231 cells. Student’s t-test P-values are shown (n=3). Error bars: s.d. (c) Immunoblots of TWF1 (green band, top panel), VIM (green band, middle panel), SNAI2 (green band, bottom panel) and β-actin (red band, top panel) with protein lysates of MDA-MB-231 cells 36 h after transfections of mock control, scrambled (Scr) and oligo miRNA-30c, respectively. (d) Effects of miR-30c on cell morphology and F-actin. Top panels: images of MDA-MB-231 cells after miR-30c transfections. Top left panels: bright field (BF) images of cells (X200, black scale bars, 10 μm). Top right panels: fluorescent images (X1,000, white scale bars, 5 μm) with F-actin staining (green) and DAPI staining of DNA (blue). Bottom histogram: percentage of round cells counted in two groups, scrambled and 30c-transfected cells (n=4, counting 4 replicates of 100–150 cells). Cells were collected 36 h after transfections of scramble control (top) or miR-30c oligos (bottom). Student’s t-test P=0.043. Error bars: s.d. (e) Luciferase activity assays of Hek293T cells co-transfected with miR-30c (or scrambled control, scr) and the luciferase vector containing wild-type (WT) or mutated (mt) 3′UTR of TWF1. Mt1, 2, 3 are mutants with G/C converted to T in individual predicted binding site 1, 2, or 3, and mt123 contains all three mutated sites (see Supplementary Fig. S3). Student’s t-test P=0.0001 for WT or mt2, comparing 30c with scr control (n=5). Error bars: s.d.