Figure 4.

FGF2 regulates mesenchymal transition- and proliferation-related gene expression at the transcriptional level through SNAI1 in human primary corneal endothelial cells in vitro. A, to determine application time point of SNAI1 siRNA, human primary corneal endothelial cells were transfected with SNAI1 siRNA and total protein was isolated at indicated times (3, 7, or 14 days) post transfection. siRNA knockdown of FGF2-dependent SNAI1 protein expression was observed at up to 14 days post transfection. Keratin 12 was used as control for corneal epithelial cell contamination. B, SNAI1 siRNA knocked down FGF2-dependent SNAI1, SNAI2, ZEB1, and ZEB2 expression in human primary CEC. C, SNAI1 siRNA knockdown led to inhibition of FGF2-dependent expression of COL1A1, COL1A2, and fibronectin; however, there was no change in expression of vimentin. FGF2-dependent suppression of E-cadherin was also reversed by SNAI1 siRNA. D, SNAI1 siRNA transfection inhibited FGF2-dependent expression of CDK2 and cyclin E1 in primary human CEC. COL8A2 and β-actin were used as corneal endothelial marker and loading control, respectively. Transfection with non-targeting control siRNA did not have any effect on gene expression in all experiments. Veh C, vehicle control; Epi, corneal epithelium; NT, non-targeting control.