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

Zhiqiang Wei; Juan Cao; Xu Zhang; Di Yin; Deyu Xu; Guoyuan Lu

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

Int J Clin Exp Pathol. 2019 Jul 1;12(7):2516-2525. eCollection 2019.

Authors

Zhiqiang Wei¹, Juan Cao², Xu Zhang², Di Yin², Deyu Xu¹, Guoyuan Lu¹

Affiliations

¹ Department of Nephrology, The First Affiliated Hospital of Soochow University Suzhou, People's Republic of China.
² Department of Nephrology, Affiliated Taixing People's Hospital of Yangzhou University Taixing, People's Republic of China.

PMID: 31934078
PMCID: PMC6949584

Abstract

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, α-smooth muscle actin (α-SMA), and fibrogenesis markers Collagen I and fibronectin (FN) were examined by RT-qPCR assay. The protein levels of E-cadherin, α-SMA and Collagen I, transforming growth factor β1 (TGF-β1)/Smad3/integrin-linked kinase (ILK) pathway-related protein TGF-β1, 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-β1 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-β1 as a target gene of miR-541, and subsequent luciferase reporter assay and western blot further supported the prediction. miR-541 counter-regulated TGF-β1 expression, and inhibited the TGF-β1/Smad3/ILK pathway. Alb treatment activated the TGF-β1/Smad3/ILK pathway, while EPA inhibited the activation of the pathway. miR-541 inhibitors reversed the effects of EPA on EMT, fibrosis, and TGF-β1/Smad3/ILK pathway-related protein expression induced by Alb.

Conclusion: EPA attenuates EMT and renal fibrosis through the TGF-β1/Smad3/ILK pathway in renal epithelial cells by targeting miR-541.

Keywords: Alb; EMT; EPA; TGF-β1/Smad3/ILK signaling pathway; miR-541; renal fibrosis.