Figure 4.

miR-888 directly targets the Smad4 3′-UTR and regulates the TGF-β1/Smad-mediated epithelial-mesenchymal transition (EMT) signaling. (A) The complementary sequences between the position 1349–1369 of the wild-type or mutant human Smad4 3′-UTR and miR-888. (B, C) Dual luciferase reporter analysis showed the direct binding of miR-888 on Smad4 3′-UTR. HEK 293 cells (B) and SW620 cells (C) were respectively co-transfected with miR-888 or empty controls and the luciferase reporter construct containing the wild-type or mutant Smad4 3′-UTR. For each experiment, the results were normalized to the luciferase activity detected in the cells transfected with the empty vectors. ***P<0.001. (D, E) RT-PCR and western blot analyses of the effects of miR-888 on Smad4 expression in HEK 293 cells (D) and SW620 cells (E). GAPDH and β-actin were respectively used as internal controls for PCR analysis and western blotting. ***P<0.001. (F) Effects of miR-888 on the expression of key factors in the TGF-β1-mediated EMT signaling pathway. **P<0.01, ***P<0.001. All data are expressed as mean ± standard deviation.