Alcoholic liver disease (ALD) is a major global health problem, ranging from fatty liver to alcoholic hepatitis and cirrhosis, which may eventually lead to hepatocellular carcinoma (Gao and Bataller, 2011). It is generally accepted that oxidative stress plays a central role in the pathogenesis of ALD. After chronic and excessive consumption, alcohol may promote oxidative stress by increased reactive oxygen species (ROS) production and/or decreased antioxidant enzyme activities, leading to alcohol-induced liver injury (Wu and Cederbaum, 2009).
Recently, the role of microRNAs (miRNAs) in ALD is getting attention. miRNAs are highly conserved noncoding small RNAs that control gene expression at the posttranscriptional level via either the degradation of target mRNAs or the inhibition of translation (Bartel, 2004). To date, there are some studies related to the roles of miRNAs in ALD (Bala and Szabo, 2012). Dolganiuc and colleagues (2009) demonstrated that alcohol up-regulates 1% and down-regulates 1% of known miRNAs in the livers of alcohol-fed mice. Tang and colleagues (2008) showed that ethanol (EtOH) increases miR-212 expression in human intestinal epithelial cells. Bala and colleagues (2011) reported that chronic alcohol consumption increases miR-155 in RAW 264.7 macrophages and Kupffer cells of alcohol-fed mice. Yeligar and colleagues (2009) observed EtOH-induced miR-199 down-regulation in rat liver sinusoidal endothelial cells and human endothelial cells. However, the pathophysiological relevance of these miRNAs to alcohol-induced liver injury has not yet been determined. We hypothesized that miRNAs could target to some genes related to oxidative stress and participate in the pathogenesis of ALD. Computational searching with RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/submission.html) and Targetscan (http://www.targetscan.org/) displayed an miR-214 binding site at the 3′-UTR of either glutathione reductase (GSR) (NM_000637.3) or cytochrome P450 oxidoreductase (POR) (NM_000941.2). It provides a potential clue for investigating the effect of alcohol on oxidative stress involving miR-214.
Previous studies demonstrated that alcohol could induce oxidative stress via glutathione circulation pathway, in which GSR catalyzes glutathione disulfide (GSSG) into reduced glutathione (Han et al., 2006). So, GSR is the major antioxidant and redox regulator in cells. POR is a flavin-containing electron donor for all microsomal cytochrome P450. In mammalian cells, the microsomal P450-related electron transport system is an important source of ROS (Zangar et al., 2004). Moreover, POR may also act as an antioxidant defense enzyme through association with heme oxygenase-1 (HO-1) (Emerson and LeVine, 2000). Therefore, in this study, we focused on the GSR and POR genes essential for oxidative stress, identified the targeting of miR-214 to their 3′-UTRs, and elucidated their impact on alcohol-induced oxidative stress in liver cells.
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The involvement of oxidative stress in the pathogenesis of alcohol-induced liver injury was first proposed in the early 1960s by Di Luzio (1964) and subsequently supported in alcohol-fed rodents (Albano et al., 1996) and in patients with alcoholism (Aleynik et al., 1998; Meagher et al., 1999). Herein, we observed the increase in MDA level and the decrease in T-AOC in EtOH-treated Bel7402 and BRL cells. Moreover, the protein levels and activities of both GSR and POR in vitro and in vivo were decreased markedly under EtOH stimulation. These findings further confirmed the association of alcohol with oxidative stress and suggested the key role of GSR and POR in it. However, the mechanisms by which alcohol contributes to the depletion of these antioxidant defenses are still not completely understood.
Recently, the role of miRNAs in oxidative stress-mediated etiology is emerging. Sangokoya and colleagues (2010) showed that miR-144 directly targets nuclear factor-erythroid 2-related factor 2 and modulates the oxidative stress response. Bai and colleagues (2011) reported that miR-335 and miR-34a contribute to renal aging by suppressing antioxidative enzymes superoxide dismutase 2 and thioredoxin reductase 2. On the other hand, miRNAs per se may also be regulated by ROS. Thulasingam and colleagues (2011) revealed that miR-27a*, miR-27b*, miR-29b*, and miR-24-2* are down-regulated, whereas miR-21* is up-regulated in response to H2O2-induced oxidative stress in RAW 264.7 macrophages. To our knowledge, there are no studies on miRNAs in alcohol-induced oxidative stress in liver cells. In this work, we found that miR-214 directly bound to 3′-UTR of the GSR and POR genes, and repressed their endogenous expressions and activities in Bel7402 and BRL cells. Moreover, endogenous miR-214 expression was up-regulated, and GSR and POR were down-regulated after EtOH exposure in vitro and in vivo. In addition, overexpression of miR-214 decreased the GSR, POR, and T-AOC activity and increased the MDA level, and inhibition of miR-214 alleviated EtOH-induced decrease in the GSR, POR, and T-AOC activity as well as increase in the MDA level. These findings suggested miR-214 mediating down-regulation of GSR and POR might play an important role in alcohol-induced oxidative stress in live cells.
miR-214 is encoded by the miR-214 gene located in the Dynamin-3 gene intron (Lee et al., 2009) and expressed in 27 species (miRBase Sequence Database, http://www.mirbase.org). Recently, the vital role of miR-214 in human diseases is getting attention. For example, a number of studies have showed differential expression of miR-214 in human cancers. miR-214 was increased in ovarian cancer and melanoma; however, it was down-regulated in cervical cancer, breast cancer, and hepatoma. These varied levels in tumors may be due to the different target genes such as PETN (Yang et al., 2008), TFAP2C (Penna et al., 2011), GALNT7 (Peng et al., 2012), plexin-B1 (Qiang et al., 2011), Ezh2 (Derfoul et al., 2011), HDGF (Shih et al., 2012), and XBP-1 (Duan et al., 2012). miR-214 was also reported to be up-regulated in human monocytes exposed to DAMPs (Unlu et al., 2012) or AGEs (Li et al., 2011) and to contribute to inflammatory response via adenosine A2A receptor (Heyn et al., 2012). Besides, miR-214 was observed to be increased significantly in renal injury (Denby et al., 2011) and liver aging (Maes et al., 2008) in rodent models. Extensive research has suggested that continued oxidative stress is a common pathologic pathway for most chronic diseases including cancer, diabetes, and cardiovascular, neurological, and liver diseases. Therefore, we postulated that miR-214 could be a key posttranscriptional regulator in oxidative stress-mediated human diseases. Herein, we evaluated the impact of miR-214 in EtOH-induced oxidative stress and found that miR-214 was up-regulated in EtOH-treated liver cells in vitro and in vivo. In contrast, Dolganiuc and colleagues (2009) reported that miR-214 is down-regulated in the livers of alcohol-induced steatohepatitis mice. This inconsistency might be resulted from the difference between species (Wistar rat vs. C57BL/6), treatments (20% vs. 4.5% EtOH), or detection methods (real-time PCR vs. miRNA microarray using probe-containing chip). High expression of miR-214 down-regulated the expressions and enzyme activities of both GSR and POR via binding to the 3′-UTR of GSR and POR genes, transfection of pmiR-214 decreased the T-AOC activity and increased the MDA level, and inhibition of miR-214 alleviates EtOH-induced decrease in the GSR, POR, and T-AOC activities. This work not only revealed new posttranscriptional mechanism of GSR and POR gene, but also threw lights on the function of these enzymes in alcohol-induced oxidative stress.
The findings that the endogenous GSR was decreased significantly after EtOH treatment, in line with the decreased T-AOC and increased MDA level, confirmed the function of GSR as a vital antioxidant defense enzyme. The alcohol-engendered repression of GSR was also reported by Schlorff and colleagues (1999) who found plasma GSR activity in Fisher-344 rats is significantly decreased after EtOH ingestion. However, others reported that hepatic GSR activity is significantly higher in chronic EtOH feeding Sprague–Dawley rats (Bailey et al., 2001; Oh et al., 1998). This controversy suggested that GSR may respond to alcohol in a generic-specific and time-dependent manner. The alcohol-engendered repression of GSR exacerbates oxidative stress, whereas in the long term, increased GSR may also be a protective mechanism against EtOH-induced chronic oxidative damage.
Similar to GSR, EtOH treatment also repressed the POR, indicating an antioxidant defense role of POR in our study. POR is a ubiquitously expressed NADPH oxidoreductase that reduces a number of important cellular electron acceptors including the cytochrome P450 enzymes those are considered as 1 of the largest sources of intracellular ROS (Mishin et al., 2010). On the other hand, POR is required for HO-1 function and favors an antioxidant state (Emerson and LeVine, 2000). It is reported that EtOH regulates HO-1, which protects the liver from alcohol-induced liver injury (Yeligar et al., 2010). Therefore, we postulated that the alcohol-induced oxidative stress observed in our study might in part result from the repression of POR and in turn the disfunction of HO-1.
Taken together, we reported for the first time that alcohol repressed the GSR and POR expression via up-regulation of miR-214 and in turn induced oxidative stress in liver cells. The regulation mechanism of miRNAs in oxidative stress pathway will be helpful to provide novel therapeutic targets to ameliorate alcohol-induced liver injury.