EPA attenuates epithelial-mesenchymal transition and fibrosis through the TGF-β1/Smad3/ILK pathway in renal tubular epithelial HK-2 cells by up-regulating miR-541

Background: It was reported that eicosapentaenoic acid (EPA) could prevent tubulointerstitial injury in kidney. EPA could also inhibit the epithelial-mesenchymal transition (EMT) of HK-2 cells stimulated by albumin (Alb) in vitro. However, the regulating molecular mechanism of EPA remains to be elucidated. Methods: An immortalized human proximal tubular cell line (human kidney-2 (HK-2) cells) was used in all experiments. MTT assay was employed to determine the effect of Alb or EPA on the cell viability of HK-2 cells. The miR-541 expression, the mRNA levels of EMT markers E-cadherin, alpha-smooth muscle actin (alpha-SMA), and fibrogenesis markers Collagen I and fibronectin (FN) were examined by RT-qPCR assay. The protein levels of E-cadherin, alpha-SMA and Collagen I, transforming growth factor (TGF-beta 1)/Smad3/integrin-linked kinase (ILK) pathway-related protein TGF-beta 1, pSmad2/3, Smad7 and ILK were measured by western blot. Enzyme-linked immunosorbent assay (ELISA) was performed to detect FN expression. The target relationship between miR-541 and TGF-beta 1 was confirmed by bioinformatics, luciferase reporter assay and western blot. Results: Low doses of Alb had no effect on the cell viability of HK-2 cells, while EPA repressed the cell viability of HK-2 cells in a concentration-dependent manner. EPA could inhibit EMT and fibrosis and increase the miR-541 expression of HK-2 cells exposed to Alb. Interestingly, introduction of miR-541 effectively abolished the EMT and fibrosis of HK-2 cells stimulated by Alb. Bioinformatics analysis predicted TGF-beta 1 as a target gene of miR-541, and subsequent luciferase reporter assay and western blot further supported the prediction. miR-541 counter-regulated TGF-beta 1 expression, and inhibited the TGF-beta 1/Smad3/ILK pathway. Alb treatment activated the TGF-beta 1/ Smad3/ILK pathway, while EPA inhibited the activation of the pathway. miR-541 inhibitors reversed the effects of EPA on EMT, fibrosis, and TGF-beta 1/Smad3/ILK pathway-related protein expression induced by Alb. Conclusion: EPA attenuates EMT and renal fibrosis through the TGF-beta 1/Smad3/ILK pathway in renal epithelial cells by targeting miR-541.