MicroRNA-195 Activates Hepatic Stellate Cells In Vitro by Targeting Smad7.

Li-Ying Song,Wei-Jin Fang,Shi-Kun Liu,Yu-Tao Ma,Chun-Jiang Wang,Cui-Fang Wu

doi:10.1155/2017/1945631

Abstract

Background and Aim Aberrant activation of the TGF-β1/Smad pathway contributes to the activation of hepatic stellate cells (HSCs). MicroRNA-195 has been shown to regulate the activation of HSCs. The aim of this study was to investigate the role of miRNA-195 in HSCs activation. Methods A liver fibrotic rat model induced by diethylnitrosamine was established. Dual luciferase reporter assays were performed to verify that Smad7 was the target of miRNA-195. The expression levels of miR-195, Smad7, and α-SMA in HSC-T6 transfected, respectively, with miR-195 mimic, inhibitor, or control were measured by qRT-PCR. The protein expression of Smad7 was detected by Western blot analysis. Results Enhanced miR-195 and decreased Smad7 were observed in diethylnitrosamine-induced liver fibrotic rats (P < 0.05). Dual luciferase reporter assays showed that the miR-195 mimic significantly suppressed the luciferase activity of a reporter plasmid carrying the binding site of miR-195 on the 3′UTR of Smad7 (P < 0.05). The miR-195 mimics activated HSCs, further elevated miR-195 and α-SMA (P < 0.01), and reduced the Smad7 level (P < 0.05). The miR-195 inhibitors blocked the activation of HSCs, reduced the expression of miR-195 and α-SMA (P < 0.01), and upregulated the expression of Smad7 (P < 0.05). Conclusion Collectively, we demonstrated that miRNA-195 activated HSCs by targeting Smad7.

Highlights

The prevalence of liver fibrosis has increased due to hepatitis B virus, alcohol toxicity, inflammation, and diabetes via increased synthesis and decreased degradation of extracellular cell matrix (ECM) in hepatocytes [1,2,3]
HSCT6 was treated with Transforming growth factorβ1 (TGF-β1) and miR-195 mimic or miR-195 inhibitor, and we examined their effects on miR-195, α-Smooth muscle actin (α-SMA), and Smad7 expression
Our research showed that miR-195 inhibitor did not reduce the level of α-SMA or elevate the level of Smad7 at 72 h, we conclude that miR-195 inhibitor could not regulate the α-SMA and Smad7 expression with a time-dependent manner, on one aspect, the transfection reagents play the best effect at different time in different cells, and on the other aspect, transforming growth factor- (TGF-)β1 could significantly reduce the Smad7 level and increase α-SMA level at a time-dependent manner which may counteract the transfection reagents role in 72 h

Summary

Introduction

The prevalence of liver fibrosis has increased due to hepatitis B virus, alcohol toxicity, inflammation, and diabetes via increased synthesis and decreased degradation of extracellular cell matrix (ECM) in hepatocytes [1,2,3]. This condition is associated with an increased risk of diseases, such as hepatocellular carcinoma (HCC), which is a leading cause of death worldwide [4]. Most of the studies have shown that miRNAs regulate the activation of HSCs in liver fibrosis. We demonstrated that miRNA-195 activated HSCs by targeting Smad

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