miR-374a/Myc axis modulates iron overload-induced production of ROS and the activation of hepatic stellate cells via TGF-β1 and IL-6

https://doi.org/10.1016/j.bbrc.2019.05.152Get rights and content

Highlights

  • MiR-374a levels were decreased in liver cells under FAC-induced iron overload.

  • miR-374a targets Myc to modulates L02 secretion of TGF-β1 and IL-6.

  • miR-374a/Myc inhibits iron-induced cytokine release to suppress the activation of hepatic stellate cells.

Abstract

The transformation of hepatic stellate cells (HSCs) to activated myofibroblasts plays a critical role in the progression of hepatic fibrosis, while iron-catalyzed production of free radical, including reaction and active oxygen (ROS), and activation and transformation of HSC into a myofibroblasts has been regarded as a major mechanism. In the present study, we attempted to investigate the mechanism of iron overload in hepatic fibrosis from the perspective of regulating HSC activation via oxidative stress and miR-374a/Myc axis. FAC stimulation significantly increased ROS production and TGF-β1 and IL-6 release dose-dependently in hepatocytes. miR-374a could target Myc, a co-transcription factor of both TGF-β1 and IL-6, to negatively regulate Myc expression; FAC stimulation significantly suppressed miR-374a expression, whereas the suppressive effect of FAC stimulation on miR-374a expression could be reversed by ROS inhibitor NAC, indicating that miR-374a could be modulated by iron overload-induced ROS. Via targeting Myc, miR-374a overexpression significantly reduced FAC-induced increases in TGF-β1 and IL-6 levels within L02 cells, whereas the effects of miR-374a overexpression were significantly attenuated via Myc overexpression. Finally, miR-374a overexpression attenuated FAC-induced activity of HSCs by decreasing α-SMA and Collagen I levels whereas Myc overexpression enhanced FAC-induced activity of HSCs by increasing α-SMA and Collagen I levels; the effects of miR-374a overexpression could also be significantly reversed by Myc overexpression, indicating that miR-374a suppresses the activation of HSCs by inhibiting Myc to reduce FAC-induced increases in TGF-β1 and IL-6 release. In conclusion, we demonstrate a novel mechanism of miR-374a/Myc axis modulating iron overload-induced production of ROS and the activation of HSCs via TGF-β1 and IL-6.

Introduction

Hepatic fibrosis, a worldwide healthy threaten with high morbidity and mortality [1], represents a reversible wound healing response to cell injury factors, including alcohol, drugs, viral infection, metabolites, or autoimmune imbalances, with the characteristics of extra-cellular matrix (ECM) accumulation and hepatic damage [2]. Hepatic stellate cells (HSCs) exerts a critical effect on hepatic fibrotic pathology. After liver fibrosis injury, HSC can trans-differentiate into an activated myofibroblast phenotype via an increase in α-smooth muscle actin (α-SMA) and Collagen I, thus promoting hepatic fibrotic development [[3], [4], [5]].

Studies have shown that various chronic liver diseases leading to liver fibrosis are associated with disorders of liver iron metabolism. Liver iron overload can be found in a variety of chronic hepatic diseases. Iron is thought to exert an effect on the promotion of liver fibrosis by various mechanisms, including iron-catalyzed production of free radical and lipid peroxidation and activation and transformation of HSC into a myofibroblastic cell [6]. Moreover, iron overload generates hydroxyl radicals (·OH) through reaction and active oxygen (ROS), superoxide anion (O2) and hydrogen peroxide (H2O2) in hepatocytes [6]; ROS is also a profibrotic factor [[7], [8], [9]]. In addition to oxidative stress, iron directly contributes to inflammatory responses via inducing nuclear factor κB, thereby increasing tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and transforming growth factor beta (TGFβ) [[10], [11], [12]]. Besides, the iron deposition could directly activate HSC. Therefore, iron overload might stimulate the oxidative stress response in hepatocytes to promote the activation of HSCs; developing an in-depth understanding of the underlying mechanism might provide novel strategies for the treatment of hepatic fibrosis.

miRNAs are small, endogenous and non-coding RNAs, which inhibit the translation or induce the degradation of mRNAs to modulate target gene expression [13]. There is growing evidence that expression profiles of miRNAs demonstrate a considerable overlap within fibrosis diseases. It has been proved that miRNAs play a role in HSC activation. For instance, a significantly higher expression of let-7 family members is observed in HSCs of bile duct ligation (BDL) animals whereas a lower expression of miR-150, -187, −194 and −207 is observed [14]; miR-150 and miR-194 overexpression within human HSCs could not only suppress the proliferation of HSCs but also prevent their transdifferentiation [14]. miR-150 and miR-94 could suppress c-Myb and Rac-1, and these two proteins participate in pathways which promote the occurrence and the development of liver fibrosis. Several miRNAs bind to a variety of genes which contribute to the development of hepatic fibrosis; likewise, some miRNAs usually modulate one single abnormally expressed gene. In summary, miRNAs might be involved in iron overload-induced oxidative stress and related HSC activation.

In the present study, the effects of ferric ammonium citrate (FAC) on ROS production and TGF-β1 and IL-6 expression and release could be examined in hepatocytes L02 cells. Since Myc has been identified as a co-transfection factor of both TGF-β1 and IL-6 via Harmonizome online database, next, miRNAs that might be downregulated by hypoxia and might target Myc were analyzed while miR-374a could be selected. The putative binding of miR-374a to Myc, the involvement of miR-374a in Myc expression, as well as the dynamic effects of miR-374a and Myc upon TGF-β1 and IL-6 levels were examined upon FAC stimulation. Finally, we investigated whether iron overload-induced ROS could activate HSCs via miR-374a/Myc axis. These observations indicate a new mechanism for iron overload from the perspective of regulating HSC activation via oxidative stress and miR-374a/Myc axis.

Section snippets

Cell line and cell transfection

L02 cell line was obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China) and cultured in Minimum Essential Medium (MEM Eagles with Earle's Balanced Salts, MEM-EBSS) supplemented with 10% FBS. Cells were cultured at 37 °C in 5% CO2.

Myc overexpression was conducted by the transfection of Myc-overexpressing vector (Shanghai Sangon Biological and Technological Company, Shanghai, China). The overexpression or inhibition of miRNA was conducted by transfecting the target cell

Iron overload induces an oxidative stress response in hepatocytes L02 cell line

To validate the involvements of iron overload in oxidation-antioxidation function in hepatocytes, we exposed L02 cells to FAC stimulation (0, 50, 100, and 150 μM) and examined the ROS production and TGF-β1 and IL-6 release. Fig. 1A showed that FAC stimulation significantly induced the increases in ROS production dose-dependently. Consistently, TGF-β1 and IL-6 expression and release could be significantly enhanced by FAC stimulation dose-dependently (Fig. 1B and C). In summary, iron overload

Discussion

Herein, FAC stimulation significantly increased ROS production and TGF-β1 and IL-6 release dose-dependently in hepatocytes. miR-374a could target Myc, a co-transcription factor of both TGF-β1 and IL-6, to negatively regulate Myc expression; FAC stimulation significantly suppressed miR-374a expression, whereas the suppressive effect of FAC stimulation on miR-374a expression could be reversed by ROS inhibitor NAC, indicating that miR-374a could be modulated by iron overload-induced ROS. Via

Acknowledgement

This study was supported by the scientific research program of Health and Family Planing Commission of Hunan Province (C20180721), scientific research program of Health Commission of Hunan Province (B2019061) and Innovation Project of Science and Technology Department of Hunan Province (2017SK50518).

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