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Keywords:

  • Glutathione Reductase;
  • Cytochrome P450 Oxidoreductase;
  • microRNA-214;
  • Alcohol;
  • Oxidative Stress

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background

The involvement of oxidative stress in the pathophysiological process of alcohol-induced liver injury has been studied for decades. However, the role of microRNAs (miRNAs) targeting to oxidative stress genes in the pathogenesis of alcohol-induced liver injury has not yet been determined. The aim of this study was to identify the targeting of miR-214 to both glutathione reductase (GSR) and cytochrome P450 oxidoreductase (POR) genes and elucidate their impact on alcohol-induced oxidative stress in liver cells.

Methods

The miR-214 expression vector and reporter vectors of GSR and POR 3′-UTR were constructed. Human hepatoma cell (Bel7402), human embryonic kidney 293 cell (HEK293), and rat normal hepatocyte (BRL) were transfected and stimulated with ethanol (EtOH). Wistar rats were fed with EtOH for 4 weeks. The GSR and POR protein levels were detected by Western blot, and their activities were measured using the spectrophotometric method. The miR-214 expression was detected by real-time PCR. The index of oxidative stress including the total antioxidant capacity (T-AOC) and malondialdehyde (MDA) level was detected by commercial kits.

Results

miR-214 bound specifically to the GSR and POR 3′-UTR and repressed the expressions and activities of both GSR and POR. EtOH up-regulated the miR-214 expression, down-regulated the GSR and POR protein levels and activities, and induced the oxidative stress in human and rat liver cells. EtOH-fed Wistar rats further confirmed that alcohol up-regulates the miR-214 expression in liver and repressed both GSR and POR in vivo.

Conclusions

These findings demonstrated a new mechanism by which the alcohol repressed the GSR and POR expression via up-regulation of miR-214 and in turn induced oxidative stress in liver cells.

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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Plasmid Construction

The human miR-214 precursor (pri-miR-214) was amplified by PCR using the primers listed in Table 1 and cloned into the pcDNA3.1(+) (Invitrogen, Grand Island, NY) to generate the miR-214 expression vector (pmiR-214). The 3′-UTRs of GSR and POR were inserted into the pGL3-promotor vector (Promega, Madison, WI) to generate pGL3-GSR and pGL3-POR. Mutations were introduced in the miR-214 binding site in pGL3-GSR-mut and pGL3-POR-mut. The authenticity and orientation of all the constructs were confirmed by DNA sequencing.

Table 1. Primers and Oligonucleotides
NameSequence (5′–3′)
  1. GSR, glutathione reductase; POR, P450 oxidoreductase.

pri-miR-214-F5′-GAATTCTGTTACGCAAATTATCCATGTTAG-3′
pri-miR-214-R5′-TCTAGAGCCCCTCATTTTGGTTGTA-3′
GSR-3′-UTR-F5′-TATAGCTCTAGACGAAGGCAGACTTTGACAACAC-3′
GSR-3′-UTR-R5′-TATAGCTCTAGACTGAAATAGCACCACCGCACTCC-3′
POR-3′-UTR-F5′-TCTAGACTCCTGGGTGTGTTTGGCTT-3′
POR-3′-UTR-R5′-TCTAGACCAAGGGCCAGAGGTTATTT-3′
Inhibitor-miR-2145′-ACUGCCUGUCUGUGCCUGCUGU-3′
Inhibitor-NC5′-CAGUACUUUUGUGUAGUACAA-3′
RT primer5′-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACCTGCCTG-3′
miR-214 forward5′-CGGCGGACAGCAGGCACAGAC-3′
miR-214 reverse5′-GTGCAGGGTCCGAGGT-3′
U6-forward5′-CTCGCTTCGGCAGCACA-3′
U6-reverse5′-AACGATTCACGAATTTGCGT-3′

Cell Culture, EtOH Treatment, and Transfection

The human hepatoma cells (Bel7402) were grown in RPMI 1640, and the human embryo kidney HEK293 cells and the rat liver cells (BRL) were grown in DMEM, supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin, in a humidified atmosphere of 95% air and 5% CO2 at 37°C. EtOH treatment was performed at 100 mM for 0, 0.5, 1, 12, 24, and 36 hours, respectively, or at the concentration of 0, 50, 100, 150, 200, and 250 mM for 24 hours. Transfection was performed using the Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

Luciferase Reporter Assay

HEK293 and Bel7402 cells were cotransfected with 600 ng of pmiR-214 or 100 nM of miR-214 inhibitor (Ambion, Grand Island, NY) and 200 ng of pGL3-GSR or pGL3-POR reporter constructs or mutants. The Firefly and Renilla luciferase activities were measured consecutively using Dual-Glo Luciferase Assays (Promega) with a Lumat LB9507 luminometer (Berthold Technologies, Bad Wildbad, Germany).

Oxidative Stress Measurements

Total antioxidation capacity (T-AOC) and lipid malondialdehyde (MDA) level were measured using commercial kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocols in a microplate reader (Bio-Rad, Philadelphia, PA).

Animal Care and Experiment Protocol

Male Wistar rats, weighing 200 to 250 g, were randomly divided into 6 groups and treated with 20% EtOH (2 g/kg, n = 15) for 1 day and 1, 2, 3, and 4 weeks via orogastric tube or 0.9% NaCl (n = 6, control). The rats were housed individually at a temperature of 23 to 25°C and were maintained on a 12L:12D photoperiod. The rats were sacrificed 30 minutes after administration, and livers were isolated in ice phosphate buffered saline, immersed in liquid nitrogen immediately, and stored at −80°C. All animal care and experiments were followed the Guiding of Principles for the Care and Use of Animals in the Field of Physiological Science.

Western Blot

Total protein was separated and transferred to a PVDF membrane. Then, the membrane was blocked for 2 hours and incubated with antibodies against GSR (Abcam, Cambridge, MA), POR (Millipore, Billerica, MA; and Abcam), hepatoma-derived growth factor (HDGF; Abcam), or GAPDH antibody (Proteintech Group Inc., Chicago, IL) overnight at 4°C. Immunoreactive bands were visualized using ECL plus chemiluminescence reagent (Thermo, Rockford, IL) and analyzed with ECL chemiluminescence detection system (Bio-Rad).

Detection of GSR and POR Activities

The GSR activity was measured via NADPH consumption against time using a spectrophotometer at 340 nm. The reaction mixture contained 1 mM GSSG and 0.1 mM NADPH (Roche, Basel, Switzerland) in 0.1 M phosphate buffer (pH 7.0). The POR activity assay was performed applying cytochrome c reduction and monitored against time at 550 nm. The assay system contained an NADPH regeneration system, 5 μM NADPH, 100 μM cytochrome c (Sigma, St. Louis, MO), 2 mM glucose-6-phosphate (Sigma), and 3 units of glucose-6-phosphate dehydrogenase (Sigma) in 1 ml volume.

Real-Time PCR

For the quantitative analysis of mature miR-214, 2 μg of RNA was reversely transcribed using an miRNA-specific stem–loop primer (Table 1). Real-time PCR was performed using the SYBR PCR Master Mix (Applied Biosystems, Foster, CA) on the Applied Biosystems 7500 Fast Real-time PCR System. U6 snRNA was amplified as a normalization control. The relative expression level was calculated using comparative Ct method.

Statistics Analysis

All data were expressed as mean ± SD from 3 experiments. Statistics analysis was carried out using the 2-tailed, unpaired Student's t-test. < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

MiR-214 Binds to the 3′-UTR of GSR and POR Genes

According to the computational prediction that both POR and GSR 3′-UTRs contain sequences complementary to miR-214 (Fig. 1A,B), we initially identified the miR-214 binding to 3′-UTR of POR and GSR. We constructed the miR-214 expression vector and the 3′-UTR reporter constructs. As shown in Fig. 1C,D, transfection of pmiR-214 in HEK293 cells decreased significantly the luciferase activities of pGL3-GSR (28%, = 0.004) and pGL3-POR (30.9%, = 0.006) compared with the pcDNA3.1(+) control vector (pc3.1), but not for their mutant constructs. Similar results were observed in Bel7402 cells (22.5% for pGL3-GSR, = 0.003; 20.8% for pGL3-POR, p = 0.004). Furthermore, repressions of pGL3-GSR and pGL3-POR by miR-214 were verified through cotransfection of miR-214 inhibitor (Inh-214), which ameliorated the decrease in luciferase activities compared with the control inhibitor (Inh-NC) (Fig. 1E,F). These findings confirmed the specific binding of miR-214 to the GSR and POR 3′-UTR.

image

Figure 1. Identification of miR-214 binding to the 3′-UTR of glutathione reductase (GSR) and P450 oxidoreductase (POR) genes. (A, B) Schematic representations of the miR-214 binding sites in the 3′-UTR of GSR and POR, and the mutant constructs. (C, D) Overexpression of pmiR-214 decreased the luciferase activity of pGL3-GSR and pGL3-POR in both HEK293 and Bel7402 cells. (E, F) Cotransfection of the miR-214 inhibitor (Inh-214) in the HEK293 cells alleviated the suppression of miR-214 on pGL3-GSR and pGL3-POR. Data are mean ± SD for 3 independent experiments. pc3.1, pcDNA3.1(+) control vector; Inh-NC, control inhibitor. **< 0.01.

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miR-214 Down-Regulates Endogenous GSR and POR

We next investigated the effect of miR-214 on endogenous GSR and POR in human and rat liver cells. Western blot demonstrated that the protein levels of both GSR and POR were decreased significantly after pmiR-214 transfection in Bel7402 (30.4% for GSR and 39.4% for POR) as well as in BRL (49.7% and 58.6% for POR; Fig. 2A,B), and the down-regulation was abolished by cotransfection of the miR-214 inhibitor. Similarly, overexpression of miR-214 also led to the significant decrease for the activities of both GSR (Fig. 2C) and POR (Fig. 2D), which could be alleviated by miR-214 inhibition. Therefore, these results indicated that miR-214 repressed the expressions and activities of both GSR and POR via targeting the GSR and POR 3′-UTR.

image

Figure 2. miR-214 down-regulates endogenous glutathione reductase (GSR) and P450 oxidoreductase (POR) in Bel7402 and BRL cells. Up lanes were proteins in Bel7402, and down lanes were proteins in BRL. (A, B) The GSR and POR protein levels were decreased after transfection of pmiR-214, which could be alleviated by cotransfection of miR-214 inhibitor (Inh-214). (C, D) The GSR and POR activities were decreased when overexpression of miR-214, which could be alleviated by Inh-214. Data are mean ± SD for 3 independent experiments. pc3.1, pcDNA3.1(+) control vector; Inh-NC, control inhibitor. *< 0.05, **< 0.01.

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EtOH Up-Regulates the miR-214 Expression

To address whether miR-214 is influenced by alcohol, we detected the expression of endogenous miR-214 in Bel7402 and BRL cells after EtOH exposure by real-time PCR. As shown in Fig. 3 the miR-214 expression was up-regulated by EtOH in a time- and concentration-dependent manner. Therefore, we speculated that the EtOH-induced miR-214 might play an important role in the impact of EtOH on GSR and POR expression.

image

Figure 3. Ethanol (EtOH) up-regulates the miR-214 expression in liver cells. The time gradient of 0, 0.5, 1, 12, 24, 36 hours with 100 mM EtOH, and the concentration gradient of 0, 50, 100, 150, 200, and 250 mM for 24 hours. (A, B) miR-214 was up-regulated by EtOH in Bel7402 and BRL cells compared with untreated control. *< 0.05, **< 0.01.

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EtOH Represses GSR and POR Through miR-214

To investigate whether EtOH affects GSR and POR, we measured the endogenous expressions and activities of GSR and POR in Bel7402 and BRL cells after treatment with EtOH. HDGF that has been reported being a target of miR-214 was also detected as a positive control. As shown in Fig. 4AC, EtOH treatment decreased the GSR, POR, and HDGF protein levels in Bel7402 (66.6% for GSR; 57.9% for POR; and 64.5% for HDGF) and in BRL cells (71.1% for GSR; 68.8% for POR; and 50.4% for HDGF), which were alleviated by cotransfection of miR-214 inhibitor (Inh-214). Likewise, the activities of GSR and POR were decreased markedly under EtOH stimulation both in Bel7402 (32.5% for GSR and 38% for POR) and in BRL cells (67.2% for GSR and 56.9% for POR), which could also be alleviated by Inh-214 (Fig. 4D,E). The data suggested that miR-214 might play a vital role in the EtOH-induced depletion of antioxidant defenses through repression of both GSR and POR.

image

Figure 4. Inhibition of miR-214 alleviates ethanol (EtOH)-engendered repression of endogenous glutathione reductase (GSR) and P450 oxidoreductase (POR) in Bel7402 and BRL cells. Up lanes were proteins in Bel7402, and down lanes were proteins in BRL. (AC) The GSR, POR, and hepatoma-derived growth factor (HDGF) protein levels were significantly decreased after EtOH treatment (200 mM for 24 hours), which were alleviated by miR-214 inhibitor (Inh-214). (D, E) Both GSR and POR activities were significantly decreased under EtOH exposure, which were alleviated by Inh-214. Inh-NC, control inhibitor. Data are mean ± SD for 3 independent experiments.*< 0.05, **< 0.01.

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EtOH Up-Regulates miR-214 and Down-Regulates GSR and POR in Rats

To evaluated the role of miR-214 in EtOH-induced depression of GSR and POR in vivo, we fed rats with 20% EtOH for 1 day and 1, 2, 3, and 4 weeks and detected the liver expression of miR-214, GSR, and POR. Comparing with the control group, EtOH increased the miR-214 levels (Fig. 5E) and decreased the GSR protein levels in all groups, and the POR levels were decreased after treatment with EtOH for 3 and 4 weeks (Fig. 5A,B). EtOH also decreased the activities of both GSR and POR (Fig. 5C,D). The data verified that EtOH-induced miR-214 might play a vital role in repression of both GSR and POR in vivo.

image

Figure 5. Ethanol (EtOH) regulates the glutathione reductase (GSR), P450 oxidoreductase (POR), and miR-214 in rats. The rats were treated with 20% EtOH for 1 day and 1, 2, 3, and 4 weeks. (A) GSR expression in EtOH-treated rats was significantly decreased. (B) POR expression in rats was significantly decreased in 3 and 4 week groups. (C, D) Regulation of the activities of GSR and POR was in accordance with the protein levels. (E) miR-214 expression in EtOH-treated rat liver was significantly increased.*< 0.05, **< 0.01.

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miR-214 Promotes the Alcohol-Induced Oxidative Stress

Because both miR-214 and EtOH caused the decrease in endogenous GSR and POR, we wondered how about the status of oxidative stress in Bel7402 and BRL cells after miR-214 overexpression or inhibition and EtOH induction. We measured the oxidative stress-related index including T-AOC and MDA level and found that transfection of pmiR-214 significantly decreased T-AOC (by 58.4% in Bel7402 cells, p = 0.012; by 60.9% in BRL cells, p = 0.0001; Fig. 6A) and increased the MDA level (by 25.8% in Bel7402 cells, p = 0.007; and by 47.0% in BRL cells, p = 0.0002; Fig. 6B), both of which were totally abrogated after addition of Inh-214. These indicated that miR-214 reduced antioxidant defense capability and increased lipid oxidation in Bel7402 and BRL cells.

image

Figure 6. miR-214 promotes the alcohol-induced oxidative stress in Bel7402 and BRL cells. (A, B) miR-214 overexpression significantly decreased total antioxidation capacity (T-AOC) and increased the malondialdehyde (MDA) level, both of which were totally abrogated after addition of miR-214 inhibitor (Inh-214) in both cell lines. (C, D) Ethanol treatment significantly decreased T-AOC, and increased the MDA level, both of which were alleviated after addition of Inh-214. *< 0.05, **< 0.01.

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To further evaluate the role of miR-214 in the alcohol-induced oxidative stress in Bel7402 and BRL cells, we measured the T-AOC and MDA level, after EtOH treatment and/or miR-214 inhibition. As shown in Fig. 6C,D, EtOH significantly decreased T-AOC (by 82.0% in Bel7402 cells, p = 0.039; and by 77.9% in BRL cells, p = 0.0002) and increased MDA level (by 23.2% in Bel7402 cells, p = 0.037; and by 43.2% in BRL cells, p = 0.0002). These indicated that alcohol augmented lipid oxidation and reduced antioxidant defense capability in liver cells. Furthermore, we found that Inh-214 totally abrogated the EtOH-induced decrease in the T-AOC activity and alleviated the EtOH-induced increase in MDA level slightly. These results suggested miR-214 might promote EtOH-induced oxidative stress in live cells via down-regulation of GSR and POR.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

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.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study was supported by the “973” Project of China (No. 2009CB526401) and the National Natural Science Foundation of China (No. 81270343).

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  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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