VEGF suppresses epithelial-mesenchymal transition by inhibiting the expression of Smad3 and miR-192, a Smad3-dependent microRNA

Transforming growth factor-beta 1 (TGF-beta 1)-induced epithelial-mesenchymal transition (EMT) is one of the important cellular and molecular mechanisms involved in renal fibrosis. Smad3 and miR-192 (a Smad3-dependent microRNA) are involved in TGF-beta 1-mediated EMT. Vascular endothelial growth factor (VEGF) is a renal tubular epithelial survival factor. Therefore, in the present study, we investigated the role of Smad3 and miR-192 in the effects of VEGF on TGF-beta 1-mediated tubular EMT. A human kidney cortex (HKC) cell line stably overexpressing VEGF (HKC-SOEV) was established. The normal HKC cells and HKC-SOEV cells were treated with TGF-131 (5 mu g/1) or/and LY294002 (20 mu mol/1) for 24 and 48 h (LY294002 blocks the effect of VEGF). The protein expression of Smad2, Smad3, Smad4 and phosphorylated Smad3 (p-Smad3) were measured by western blot analysis. The expression of Smad3 and miR-192 was determined by real-time PCR. E-cadherin and alpha-smooth muscle actin (alpha-SMA) expression was detected by western blot analysis and laser scanning confocal microscopy (LSCM). TGF-beta 1 was found to induce the expression of alpha-SMA in the HKC cells. TGF-beta 1 also induced Smad3, miR-192 and p-Smad3 expression, but suppressed E-cadherin expression. However, in the HKC-SOEV cells, the expression levels of a-SMA, Smad3, miR-192 and p-Smad3 upon TGF-beta 1 stimulation were significantly reduced. In these cells, the suppressive effect of TGF-beta 1 on E-cadherin was also reduced. Importantly, treatment with LY294002 significantly-diminished the effect of VEGF. VEGF suppressed Smad3 and miR-192, and subsequently inhibited EMT induced by TGF-beta 1.