Clinics and Research in Hepatology and Gastroenterology
Original articleMicroRNA-101 suppresses liver fibrosis by downregulating PI3K/Akt/mTOR signaling pathway
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.
Section snippets
Cell culture
The JS-1 cell line, a spontaneously immortalized murine HSC, was provided by Dr. Jinsheng Guo (Division of digestive diseases, Zhongshan Hospital, Fudan University, Shanghai, China). The spontaneously immortalized human HSC cell line LX-2 was a gift from Dr. Scott L. Friedman (Mount Sinai School of Medicine, New York, USA). JS-1 and LX-2 cells were cultured in Dulbecco's modified eagle medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and
MiR-101 inhibited HSC activation in vitro
TGF-β1 significantly increased the LX-2 cell viability (P < 0.01, Fig. 1A) and proliferation (indicated by upregulated EdU, P < 0.01, Fig. 1B and 1G), and it decreased the expression of miR-101 in two HSC cell lines (JS-1 and LX-2, Supplementary Fig. 1A and 1B), indicating that downregulated miR-101 was involved in the activation and proliferation of HSCs. TGF-β1 could also markedly upregulate the expression of F-actin (P < 0.01, Fig. 1C and 1H), a marker of cytoskeleton that plays a key role
Discussion
Liver fibrosis is a dynamic wound-healing process resulting from chronic hepatic insults and is characterized by excess accumulated extracellular matrix (ECM) accumulation. HSCs play a pivotal role in liver fibrosis and are regarded as the primary source of ECM [22]. TGF-β1, the key activator of HSCs and major cytokine involved in liver fibrogenesis, stimulates the transcription and release of collagen, αSMA and other ECM proteins [23]. In our study, TGF-β1 was used to induce HSC activation and
Funding
This work was supported by the National Natural Science Foundation of China (Grant No. 81270053 and 81573810) and the Natural Science Foundation of Shanghai Municipal Government (Grant No. 16ZR1437800).
Author contributions
Yan-yan TAO and Cheng-hai LIU had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Yan-yan TAO and Cheng-hai LIU formulated the hypothesis, designed the study, supervised the experiments, reviewed the data analysis, and reviewed and revised the manuscript. Yang LEI designed the study, performed the experiments, reviewed the literature, performed the data analysis, interpreted the results, and drafted and
Disclosure of interest
The authors declare that they have no competing interest.
Acknowledgements
This manuscript was edited by Wallace Academic Editing.
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