Elsevier

Cellular Signalling

Volume 26, Issue 11, November 2014, Pages 2381-2389
Cellular Signalling

MicroRNA-200a controls Nrf2 activation by target Keap1 in hepatic stellate cell proliferation and fibrosis

https://doi.org/10.1016/j.cellsig.2014.07.016Get rights and content

Highlights

  • Keap1/Nrf2 pathway plays a key role in HSC proliferation and fibrosis.

  • miRNA-200a regulates the Keap1/Nrf2 pathway in HSC activation and fibrosis.

  • miRNA-200a regulation of Keap1 results in the Nrf2 pathway activation.

  • Keap1 is a novel therapeutic target for HSC activation and fibrosis.

Abstract

Hepatic fibrosis is a common final pathological process in the progression of liver disease, which is primarily due to oxidative stress. Nrf2 is known to coordinate induction of genes that encode antioxidant enzymes. Moreover, Nrf2 expression is largely regulated through the association of Nrf2 with Keap1, which results in cytoplasmic Nrf2 degradation. Conversely, little is known concerning the regulation of Keap1 expression. Although the function of miRNA-200a controls Keap1 gene expression has been discussed in many cancers and fibrotic diseases, its role in hepatic fibrosis is still poorly understood. By using miRNA mimic, we observed miRNA-200a silencing in activated hepatic stellate cell and demonstrated that upon re-expression, miRNA-200a targets the Keap1, and leading to Keap1 mRNA degradation. We find that treatment with miRNA-200a mimics, restored miRNA-200a expression and reduced Keap1 levels. This reduction in Keap1 levels corresponded with Nrf2 nuclear translocation and activation of Nrf2-dependent NQO1 gene transcription. Moreover, we found that Nrf2 activation inhibited the TGF-β1-independent growth of hepatic stellate cell. Finally, our study demonstrates that miRNA-200a regulates the Keap1/Nrf2 pathway in hepatic stellate cell and fibrosis, and we find that epigenetic therapy can restore miRNA-200a regulation of Keap1 expression, therefore reactivating the Nrf2-dependent antioxidant pathway in liver fibrosis.

Introduction

Hepatic fibrosis is a common pathological process resulted from various chronic hepatic injuries, which is characterized by an increase of extracellular matrix (ECM) deposition in the Disse's space and the imbalance between synthesis and degradation of ECM [1], [2]. During the liver fibrosis process, hepatic stellate cells play a key role based on their ability to undergo activation following liver injury of any cause [3]. HSC activation has been recognized to be responsible for most of the excess extracellular matrix (ECM) observed in chronic liver fibrosis [4], [5]. HSC activation is triggered by different inflammatory cytokines, of which TGF-β1 is recognized as the main profibrogenic mediator [6]. The pathogenesis of hepatic fibrosis as well as the role of oxidative stress and inflammation therein is well established and, accordingly, blocking or retarding the chain reactions of oxidation and inflammation process could be promising therapeutic strategies for prevention and treatment of liver injury and fibrosis [7].

During recent years, research has focused on oxidative stress mechanisms involved in liver fibrosis [8], [9]. Increasing evidence has demonstrated that oxidative stress promotes HSC activation and collagen production and plays an important role in the pathogenesis of liver fibrosis [10], [11], [12]. Nuclear factor-erythroid-2-related factor 2 (Nrf2) is an important transcription factor responsible for inducing phase II detoxifying and antioxidant enzymes [13]. Nrf2 binds to antioxidant response elements (ARE) within promoter regions of Nrf2-regulated genes and activates gene transcription [14]. Nrf2 regulates the expression of numerous detoxifying enzymes, including glutathione S-transferases (GSTs), NQO1 (NAD(P)H-quinone oxidoreductase 1), and glucuronosyl transferases, thus protecting against carcinogen-induced DNA damage [15], [16]. Functional activity of Nrf2 strongly depends on its regulation by the Keap1 (Kelch-like ECH-associated protein 1) adaptor protein [17]. Keap1 binds to and sequesters Nrf2 in the cytoplasm, thus leading to Nrf2 ubiquitination by Cul3 and proteasomal degradation [18]. Inactivation of Keap1 releases Nrf2, resulting in Nrf2 nuclear translocation and subsequent activation of cytoprotective gene transcription [19].

What's more, microRNAs are endogenous small (18–22 nt) non-coding RNAs that have important roles in a wide range of biological processes, including cell proliferation, and differentiation [20], [21]. They are largely known to base pair with the 3′-UTR of target mRNAs, downregulating their stability and translation to regulate gene expression [22]. Specific contribution of selected miRNAs in HSC has been described, recent studies demonstrated that several miRNA-200a induce proliferation or migration of myofibroblastic-activated HSC [23]. Among miRNAs dysregulated in liver fibrosis, the miRNA-200 family is often found down-regulated and subject to aberrant epigenetic silencing [23]. Furthermore, other studies suggest that miRNA-200a regulates Keap1 expression by binding to the Keap1 mRNA 3′-UTR in breast cancer cells [24]. However, whether miRNA-200a controls Keap1 gene expression in hepatic fibrosis is still unknown. We now demonstrate that miRNA-200a contributes to dysregulation of Keap1 and interferes with the Keap1/Nrf2 pathway. Treatment of HSC with miRNA-200a mimics resulted in miRNA-200a restoration and Keap1 down-regulation, which ultimately led to reactivation of the Nrf2-dependent antioxidant pathways. These results might help in developing effective and safer therapy for liver fibrosis.

Section snippets

Materials and reagents

CCl4 was purchased from Shantou Xilong Chemistry Plant (Shantou, China). Dimethyl sulfoxide (DMSO), propidium iodide (PI), and RNaseIand MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide) were purchased from Sigma Inc. (St. Louis, MO, U.S.A.). Mouse anti-α-SMA monoclonal antibodies and β-actin antibody were purchased from Boster (Wuhan, China). Rabbit anti-Nrf2 polyclonal antibody and Keap1 antibodies were purchased from Cell Signaling (Beverly, MA, U.S.A.). TGF-β1 (Peprotech,

The expression of Keap1, Nrf2 in liver fibrosis tissue and activated HSC

H&E and Masson's trichrome staining showed normal lobular architecture with central veins and radiating hepatic cords in the vehicle group (Fig. 1A). The CCl4 treatment resulted in steatosis, inflammatory infiltration, and fibrosis in the main block of experiments of 12 weeks.

The expression of Keap1, Nrf2, and α-SMA in liver fibrosis tissue and activated HSC was determined using immunohistochemistry and quantitative real-time PCR analysis (Fig. 1B; 1C). These data showed that Keap1 expression

Discussion

Liver fibrosis disease constitutes a global connection, and the medical treatments for these diseases are usually difficult to handle and have limited efficacy [26]. The activation of HSC plays a central role in liver fibrosis [27]. Therefore, it is necessary and considerable interest to find new medicines for the treatment of liver diseases. Oxidative stress is a key factor on liver fibrosis [28]. The Nrf2 antioxidant pathway can serve as an important cellular defense against oxidative stress,

Conflict of interest statement

None.

Acknowledgments

This project was supported by Anhui Provincial Natural Science Foundation (1308085MH117, 1408085MH175). This project was also supported by the National Science Foundation of China (NO 81273526).

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