Inhibition of Integrin-Linked Kinase via a siRNA Expression Plasmid Attenuates Connective Tissue Growth Factor-Induced Human Proximal Tubular Epithelial Cells to Mesenchymal Transition

Abstract

Background: Increasing evidence suggests that connective tissue growth factor (CTGF) is involved in the epithelial-to-mesenchymal transition (EMT). The exact intracellular events that drive this process, however, are not fully understood. In this study, we investigated the role of integrin-linked kinase (ILK) in mediating CTGF-induced EMT. Methods: The expression of α-smooth muscle actin (α-SMA) and E-cadherin upon the stimulation by recombinant human CTGF (rhCTGF) in cultured human tubular epithelial cell line (HK-2) was detected by real-time RT-PCR and Western blot. Subsequently, the role of ILK was determined by using ILK siRNA. Results: rhCTGF increased the mRNA expression of α-SMA significantly in a dose- and time-dependent manner, while E-cadherin mRNA decreased in a dose- and time-dependent manner. α-SMA protein was up-regulated after stimulation by 5 ng/ml CTGF for 96 h, and increased further after stimulation by 50 ng/ml. An immunocytochemical study showed that α-SMA was initially detectable at 48 h, and increased further at 72 h, while there was almost no α-SMA immunostaining observed in the control group at the same time point. E-cadherin protein was also down-regulated in a dose-dependent manner. Transfection of HK-2 cells with ILK-siRNA significantly attenuated rhCTGF-induced α-SMA induction and E-cadherin repression. Conclusion: Our study suggested that ILK mediated the effect of EMT in proximal tubular epithelial cells stimulated by CTGF.