MicroRNA-101 suppresses liver fibrosis by downregulating PI3K/Akt/mTOR signaling pathway

Highlights

• MiR-101 has antifibrotic effect in experimental liver fibrosis.

• Down-regulating PI3K/Akt/mTOR signaling pathway is involved in the antifibrotic mechanisms of miR-101.

• The roles of miR-101 in liver fibrosis suggest that it could be a potential therapeutic target for liver fibrosis.

Summary

Background

MicroRNA-101 (miR-101) is markedly downregulated in both hepatitis B virus-related liver cirrhosis and hepatocellular carcinoma (HCC). In this study, we aimed to investigate the effect and mechanism of miR-101 on hepatic stellate cell (HSC) activation and liver fibrosis.

Materials and methods

HSC LX-2 was treated with TGF-β1 and with or without miR-101 mimics. LX-2 vitality and proliferation, the expression of F-actin and mRNAs for α-SMA, collagen 1α1 (Col 1α1), and connective tissue growth factor 2 (CCN2) were measured. A 6-week intraperitoneal injection of carbon tetrachloride (CCl4) was used to induce experimental liver fibrosis in mice, which were treated using a miR-101 negative control or miR-101 agomir from the fourth week until the end of the experiment. Liver function, hepatic hydroxyproline, liver histopathology, collagen deposition, α-SMA, type I collagen (Col I) and the protein-expressions of p-PI3K, p-Akt and p-mTOR were measured.

Results

MiR-101 significantly suppressed the increased LX-2 vitality and high accumulation of extracellular matrix (ECM) induced by TGF-β1. Exposure to CCl4 led to the impairment of liver function and disruption of normal hepatic parenchyma in mice, as well as obvious liver fibrosis indicated by elevated levels of hydroxyproline, α-SMA, and Col 1α1 in liver tissues. MiR-101 administration significantly improved liver function, relieved hepatic parenchyma damage, and reversed liver fibrosis by decreasing the accumulation of ECM components. Furthermore, miR-101 substantially downregulated the CCl4-increased p-PI3K, p-Akt, and p-mTOR in mouse liver.

Conclusions

MiR-101 has antifibrotic effects in experimental liver fibrosis, and downregulating the PI3K/Akt/mTOR signaling pathway may be one of its antifibrotic mechanisms.

Introduction

Liver fibrosis, a reversible wound-healing response against a variety of stimuli in the liver, is the major characteristic of most chronic liver diseases such as hepatitis B virus (HBV) infection and alcohol abuse. It can accelerate the progression of liver diseases to the chronic form by destroying the normal hepatic parenchyma [1] and eventually leads to liver cirrhosis, liver failure, or even primary liver cancer [1], [2]. Therefore, understanding the pathogenesis of liver fibrosis is key for the development of effective antifibrotic therapies, which have a substantial clinical impact.

MicroRNAs (miRNAs, miRs) are a subset of small endogenous noncoding RNAs, approximately 18–22 nucleotides in length, which can regulate gene expression (approximately 60% of the mammalian genome [3]) at the posttranscriptional level by targeting the 3’-untranslated region (UTR) of specific mRNAs, thereby resulting in mRNA degradation or translational repression [4]. MiRNAs play crucial roles in the homeostasis of a normal human body and in the diverse biological processes of cells [4]. Our studies have revealed significantly downregulated miR-101 in liver tissues of experimental liver fibrosis liver tissues as well as human HBV-induced liver cirrhosis and hepatocellular carcinoma (HCC) [5], [6]. Most research related to miR-101 has focused on its function in various cancers [7], [8], and little is known about the detailed mechanisms of how miR-101 is involved in liver fibrosis. One study investigated the effects of miR-101 in experimental liver fibrosis and found that miR-101 suppresses liver fibrosis by targeting the TGF-β signaling pathway [9]. Huang et al. confirmed that miR-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation [10], indicating the antifibrotic effect of miR-101 in fibrotic diseases and its potential role as a therapeutic target.

The phosphatidylinositol-3 kinase/antiapoptotic kinase (PI3K/Akt) signaling pathway exists in all cell types and is involved in a variety of cell biological processes [11], [12]. In response to a series of upstream signals, PI3K/Akt exerts biological effects by acting on its downstream signaling molecule, the mammalian target of rapamycin (mTOR), which belongs to the serine/threonine protein kinase family and plays a critical role in regulating cell proliferation, apoptosis, autophagy, and chemotherapy resistance [13], [14]. Studies have demonstrated that activation of the PI3K/Akt/mTOR signaling pathway plays a central role in liver fibrogenesis, and some treatments exert antifibrotic effects by targeting this signaling pathway [15], [16]. Bioinformatic analysis shows that miR-101 has the same binding site as the 3’UTR of mTOR mRNA [8], and miR-101 inhibits the PI3K/Akt/mTOR signaling pathway in different cancers [8], [17].

We hypothesized that miR-101 could reverse liver fibrosis by inhibiting the PI3K/Akt/mTOR signaling pathway. To demonstrate this hypothesis, we adopted both hepatic stellate cell (HSC) LX-2 and male ICR mice to induce in vitro and in vivo models of liver fibrosis, and we investigated the effects of miR-101 on experimental liver fibrosis and its regulating effects on the PI3K/Akt/mTOR signaling pathway.