microRNA-21, via the HIF-1α/VEGF signaling pathway, is involved in arsenite-induced hepatic fibrosis through aberrant cross-talk of hepatocytes and hepatic stellate cells
Graphical abstract
Introduction
Arsenic, an environmental toxicant, is widely distributed throughout the world. In many countries and regions, groundwater contaminated by arsenic is the main source of high arsenic exposure for millions of people (Naujokas et al., 2013; Huang et al., 2015). The maximum contaminant level of arsenic in drinking water stipulated by the World Health Organization (WHO) is 10 ppb (Nurchi et al., 2020). However, an epidemiological survey shows that more than 200 million people live in regions where the arsenic content in drinking water exceeds the WHO standard (Nurchi et al., 2020). As found by epidemiologic studies, chronic environmental or occupational exposure to arsenic may result in damage to multiple organs and systems, including diabetes mellitus, various cardiovascular diseases, and cancer (Nong et al., 2016; Kuo et al., 2017; Minatel et al., 2018). Therefore, chronic arsenic poisoning caused by drinking arsenic-containing water is a concern for public health.
Liver, considered to be the main organ for arsenic metabolism, is a target organ of arsenic toxicity (Stýblo et al., 2002). During chronic exposure to arsenic, various types of liver damage may occur, including steatosis, fibrosis, cirrhosis, and cancer. When arsenic is ingested, it is absorbed into the blood and distributed to the liver, where it is metabolized, mainly by methylation (Drobna et al., 2009). The methylation of arsenic produces a variety of products, which damage hepatocytes. As a toxicant, arsenic accumulates in hepatocytes, binds with sulfhydryl groups, and forms stable complexes, thus hindering cell respiration, division, and proliferation, and impeding cell metabolism (Yamauchi and Yamamura, 1983; Karimi et al., 2014). Further, the production of free radicals, increased lipid peroxidation, release of inflammatory factors, and DNA damage can cause liver damage (Shi et al., 2004; Ghosh et al., 2008). These adverse events start a downstream cascade, activating and initiating the transformation of hepatic stellate cells (HSCs) to myofibroblasts (MFs), releasing excessive deposition of extracellular matrix (ECM), and eventually leading to liver fibrosis (Ghatak et al., 2011). Hepatic fibrosis is a pathological stage in which the ECM accumulates slowly. Once cirrhosis is established, the opportunity for reversing this process is lost, and complications related to cirrhosis develop. The activation of HSCs is considered to be an essential factor in the formation of liver fibrosis. Although the pathogenesis of liver fibrosis induced by arsenic exposure is partially understood, its molecular regulation needs further study. Our previous research has shown that abnormal communication between hepatocytes and HSCs is involved in the activation of HSCs by arsenic (Dai et al., 2019; Li et al., 2019). Hepatocytes, the main cells for arsenic metabolism, are the most abundant cells in liver. Thus, aberrant cross-talk between hepatocytes and HSCs may be involved in arsenic-induced liver fibrosis.
MicroRNAs (miRNAs) are non-coding RNA molecules (∼22 nucleotides) that participate in the regulation of gene expression in cell proliferation and differentiation, apoptosis, and other processes (Bartel, 2009). The dysregulated expression of miRNAs is related to various diseases. In animals, miR-21 down-regulates tumor suppressor genes and promotes carcinogenesis (Bica-Pop et al., 2018). Since blocking of miR-21 has anti-fibrogenic effects, it may be a therapeutic target for kidney, lung, heart, and liver fibrosis (Thum et al., 2008; Liu et al., 2010; Zarjou et al., 2011). Potential mechanisms related to the regulation of liver fibrosis include miR-21 targeting of Smad7 and Spry1 and activation of the NLRP3 inflammasome/interleukin (IL)-1β axis to mediate angiotensin II-induced hepatic fibrosis (Ning et al., 2017). In addition, chlorogenic acid lessens liver fibrosis by inhibiting TGF-β1/Smad7 regulation by miR-21. For experimental and human nonalcoholic steatohepatitis, miR-21 inhibits Smad7 to advance fibrosis by leptin-mediated NADPH oxidase (Dattaroy et al., 2015). Moreover, miR-21 promotes proliferation of HSCs and inhibits apoptosis; its mechanism may related to the PTEN/PI3K/Akt pathway (Hao et al., 2018). Although the role of miR-21 in the development of fibrosis has been discussed, its effect in the fibrosis induced by arsenic needs further study.
Hypoxia-inducible factors (HIFs), the transcriptional regulators in response to hypoxia, consist of an oxygen-regulated HIF-α subunit (HIF-1α or HIF-2α) that, in hypoxia, dimerizes with HIF-1β (Pugh and Ratcliffe, 2003). When HIFs are activated, the target genes with CREB-cAMP response elements are transcribed (Semenza et al., 1996). Under normoxia, HIFs are hydroxylated in the oxygen-dependent degradation domain by HIF-α prolyl hydroxylase. This process regulates the binding of von Hippel Lindau protein (pVHL) tumor suppressor E3 ligase and Lys48-linked ubiquitination of HIF-1α, leading to degradation by proteasomes (Jaakkola et al., 2001). In humans, HIFs function as regulatory factors that activate target genes involved in the response to hypoxia, including proliferation, apoptosis, angiogenesis, regulation of the glucose transporter, and synthesis of glycolysis-related enzymes (Semenza, 2010). In non-alcoholic fatty liver disease, HIF-1α promotes liver fibrosis by activating the PTEN/p65 signaling pathway (Han et al., 2019). Up-regulation of HIF-1α is related to activation of HSCs and to liver fibrosis (Moon et al., 2009). The role of HIF-1α in the development of liver fibrosis is confirmed by experiments with Hif-1α hepatocyte knockout mice, which indicate that hypoxia and HIF-1α recruitment precede fibrosis, and that hepatocyte-specific silencing of Hif-1α leads to a reduction in deposition of ECM (Moon et al., 2009). Activation of HSCs is considered to be an essential event in the establishment of liver fibrosis. For isolated human HSCs, hypoxia-stimulated activation of HIF-1α promotes migration of HSC/MFs (Novo et al., 2012). Moreover, silencing of Hif-1α inhibits the migration of HSC/MFs, suggesting that HIF-1α is essential in the development of hepatic fibrosis (Novo et al., 2012). Vascular endothelial growth factor (VEGF) promotes angiogenesis (Thabut and Shah, 2010). Bioinformatics analysis confirms that, in the activation of HSCs, the VEGF pathway is the most abundant signaling pathway (Novo et al., 2007). Consistently, VEGF levels are elevated in activated HSCs (Corpechot et al., 2002). In HSCs, HIF-1α has a regulatory effect on VEGF, and pVHL/HIF-1α has a regulatory role in COX-2-mediated production of VEGF (Wang et al., 2004). Thus, the HIF-1α/VEGF signaling pathway is related to liver fibrosis. However, only limited information about the regulatory mechanism of the HIF-1α/VEGF signaling pathway in arsenic-induced hepatic fibrosis is available.
For clarifying the mechanism of arsenic-induced hepatic fibrosis, mice were exposed to arsenite, an active form of arsenic, in their drinking water, to induce hepatic fibrosis. High expression of miR-21 in hepatocytes regulated the stability of HIF-1α via pVHL, enhanced the HIF-1α/VEGF signaling pathway, and promoted the activation of HSCs. Further, studies were accomplished by use of wild-type (WT) and miR-21−/− mice exposed to arsenite in their drinking water. miR-21−/− mice showed less HIF-1α/VEGF signaling and less hepatic fibrosis than WT mice.
Section snippets
Animals
The WT mice used for breeders were purchased from the Animal Laboratory Center of Nanjing Medical University (Nanjing, China). The miR-21−/− C57BL/6 mice used for breeders were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). The offspring were generated from a breeding colony at the animal facility of Nanjing Medical University. Mice were housed in a specific pathogen-free room, maintained at a controlled temperature (23 ± 1 °C) and on successive
Chronic exposure of mice to arsenite induces hepatic damage, inflammation, and fibrosis in livers
To assess the relationship between arsenite and liver fibrosis, mice were dosed with 0, 10, or 20 ppm arsenite in drinking water for 12 months or with 20 ppm arsenite in drinking water for 0, 3, 6, or 12 months (Fig. 1A). The plasma levels of ALT and AST, circulating markers of liver damage, were elevated in mice dosed with 20 ppm arsenite (Fig. 1D). Inflammatory factors are involved in the progression of hepatic fibrosis, and there is a relationship between inflammatory cytokines and hepatic
Discussion
Environmental inorganic arsenic occurs mainly as arsenite (AsⅢ) and arsenate (AsⅤ), which are highly toxic and pose a serious threat to millions of people (Naujokas et al., 2013). For some regions, the widespread exposure of humans to arsenic is related to the drinking water and food supply (Mondal et al., 2010; Huang et al., 2015). Epidemiological studies show that liver fibrosis is closely correlated with exposure to arsenic (Young et al., 2018). Nevertheless, the molecular mechanisms for
Funding
This work was supported by the Natural Science Foundations of China (81903359, 81730089, and 81703835); Universities Natural Science Foundation of Jiangsu Province (19KJB330002); China Postdoctoral Science Foundation (2020M671548); the open project of Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment and Jiangsu Collaborative Innovation Center For Cancer Personalized Medicine, Nanjing Medical University, China (JX21817902/023); and the Priority Academic Program Development of
Credit author statement
Jing Sun: Conceptualization, Methodology, Visualization, Writing – original draft, Funding acquisition. Le Shi: Methodology, Formal analysis, Data curation, Funding acquisition. Tian Xiao and Junchao Xue: Software, Validation, Investigation. Junjie Li: Project administration, Methodology. Peiwen Wang: Project administration, Methodology. Lu Wu: Visualization, Investigation. Xiangyu Dai: Visualization, Investigation. Xinye Ni: Writing – review & editing, Supervision, Project administration.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors thank Donald L. Hill (University of Alabama at Birmingham, USA), an experienced, English-speaking scientific editor for editing.
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Authors contributed equally.