Abstract

Objectives: Liver transplantation is used to treat both patients with end-stage liver diseases and those with hepatocellular carcinoma; in Iran, these patients are commonly infected with hepatitis B and C viruses. In the present study, for the first time, we investigated the association between ACOX1 and NRF1 gene and protein expression and presence of hepatitis B virus, hepatitis C virus, and hepatocellular carcinoma in liver transplant patients in South-Central Iran.
Materials and Methods: In this cross-sectional study, we included 200 patients who were seen between 2008 and 2017 for liver transplant at the Namazi Hospital, Shiraz University of Medical Sciences (Shiraz, Iran). All patients received liver grafts from brain dead donors. Donors and recipients were unrelated. ABO compatibility blood group analyses for donor-recipient pairs were conducted. Liver transplant recipients were divided into 3 different groups: hepatitis B virus infected, hepatitis C virus infected, and presence of hepatocellular carcinoma. We also had a control group that included 30 healthy individuals. NRF1 and ACOX1 gene expression levels were evaluated using the SYBER green real-time polymerase chain reaction method. NRF1 and ACOX1 protein expression levels were evaluated using enzyme-linked immunosorbent analyses. We used SPSS software for statistical analyses (version 19.0).
Results: NRF1 gene expression was increased in all 3 liver transplant recipient groups compared with the control group (not significant, P > .05). Furthermore, ACOX1 gene expression was decreased in all patients compared with control (P > .05). However, we found ACOX1 and NRF1 protein expression to be significantly decreased in all 3 liver transplant recipients groups compared with the control group (P < .05).
Conclusions: NRF1 and ACOX1 genes and their protein expressions could affect the development of chronic liver disease.

Key words : Acyl-coenzyme A oxidase-1, Chronic liver disease, Nuclear respiratory factor 1

Introduction

Liver transplantation is a treatment for patients with end-stage liver disease.^1,2 Liver transplant is also considered for patients with end-stage cirrhosis and those with hepatocellular carcinoma (HCC).³ Liver transplant remains the best chance for long-term survival in patients with unresectable HCC.⁴ Chronic liver disease (CLD) and HCC are the leading causes of end-stage liver disease in Iran; these patients are commonly infected with hepatitis B virus (HBV) and hepatitis C virus (HCV).^5-7 The main reasons for CLD include HBV^8,9 and HCV^10,11 infections,^12,13 metabolic fatty liver disease, and alcohol use. In addition, patients with CLD are more likely to have simultaneous infections at the time of fundamental cellular imperfection, genetic predisposition, and innate immune dysfunction.¹² Both HCV and HBV infections lead to reactive oxygen species generation, increased DNA mutations, and dysfunction of the DNA repair mechanism.¹⁴

An essential factor for learning about HCC development is the identification of gene pathways and genetic networks.¹⁵ Numerous genes have been recognized in HCC tissue samples and in viral hepatitis using cell culture methods and mouse models. In humans, nuclear respiratory factor 1 (NRF1; also known as nuclear factor erythroid-2 related factor-1; NFE2L1) is located on chromosome 17q21.3 (a recessive gene).^16,17 NRF1 is a transcription factor that belongs to the CNC basic-region leucine zipper (bZIP) family.¹⁸ NRF1 has been shown to play significant roles in cellular and organ homeostasis, various pathophysiological processes, embryonic growth, organ differentiation, oxidative stress response, and hepatic lipid metabolism.¹⁹ On the other hand, NRF1 controls the induction of drug-metabolizing enzymes, which involve antioxidant response elements (AREs) similar to the NFE2-binding motif.²⁰ NRF1 also plays a role in inflammation by regulating inducible nitric oxide synthase expression in the liver. Inducible nitric oxide synthase plays a role in the creation of nitric oxide.

Previous NRF1 knockout mouse studies have shown an essential gene in hepatocyte homeostasis, pathogenesis, and cancer.²¹ Gene expression investigations have shown that reducing the expression of numerous ARE genes and the somatic inactivation of NRF1 can lead to oxidative stress in mutant hepatocytes in the liver.^22,23 NRF1 influences cell survival via keeping embryonic hepatocytes by redox balance from tumor necrosis factor and apoptosis. The reduction of NRF1 through the development of cells increases the cytotoxic effects of tumor necrosis factor in hepatocytes.²⁴ Tumor necrosis factor is the main cytokine for inflammation responses in the cell.²⁵ NRF1 deficiency leads to decreased expression of the proliferator-activated receptor-γ coactivator 1β and lipin1, and these genes are transcriptional coactivators.²⁶ Amazingly, NRF1 hepatocyte-specific disruptions will cause oxidative stress, inflammation, cell death, hepatic steatosis,²⁷ spontaneous hepatic cancer, liver tumors, steatohepatitis, and fibrosis.^16,19 Furthermore, the restricted loss of NRF1 in mouse liver leads to development of steatosis and cancer.^28,29

The acyl-coenzyme A oxidase-1 (ACOX1) gene is located on chromosome (17q25.1).¹⁶ ACOX1 is the prime and the rate-preventive enzyme of the peroxisome β-oxidation pathway.^30,31 It is mainly activated by peroxisome proliferators.³² ACOX1 contributes to production of some fatty acids, for example, polyunsaturated fatty acids and very-long-chain fatty acids.³⁰ ACOX1 is the primary enzyme for dicarboxylic fatty acid (DCA) breakdown in humans and mice, and DCA disrupts the respiratory chain activity.³³ The enzyme of ACOX1 is shown in peroxisomal fatty acid β-oxidation, and deficiency of the enzyme is related to pseudo-neonatal-adrenoleukodystrophy (a lethal, autosomal recessive disease).³⁴ Peroxisomal ACOX1 enzyme activity has been shown to contribute to cellular respiration and heat production.³⁴ Interestingly, mice with ACOX1 deficiency show severe inflammatory steatohepatitis with a high level of intrahepatic H₂O₂.³⁵ The peroxisomal ACOX1 enzyme is a leading producer of H₂O₂. ACOX1 dysfunction is associated with HCC and peroxisomal disorder.³⁶ To our knowledge, so far there are no reports on the association between ACOX1 and NRF1 and HBV and HCV infections and HCC in liver transplant patients. We thus conducted this study to find possible associations in liver transplant patients in South-Central Iran.

Materials and Methods

Patients
This cross-sectional study included 200 patients who were seen between 2008 and 2017 for liver transplant at the Namazi Hospital, Shiraz University of Medical Sciences (Shiraz, Iran). The study was approved by the Ethics Committee of Shiraz Medical University and the University of Zabol, and protocols conformed to the ethical guidelines of the 1975 Helsinki Declaration. All participants signed written informed consent.

All patients received liver grafts from brain dead donors. Donors and recipients were unrelated. ABO compatibility blood group analyses for donor-recipient pairs were conducted. Liver transplant recipients were separated into 3 different groups:
150 HBV-positive patients, 21 HCV-positive patients, and 29 patients with HCC. We also had a control group of 30 healthy individuals for comparisons.

Liver transplant recipients in all groups received immunosuppressive drugs. Recipients received cyclosporine (5.0 mg/kg of body weight/day) and tacrolimus (0.3 mg/kg of body weight/day). All patients received tests for human immunodeficiency virus (Pro-Diagnostic Bioprobes), HBV (Pro-Diagnostic Bioprobes), and HCV (Pro-Diagnostic Bioprobes) before liver transplant.

RNA extraction and cDNA synthesis
RNA was extracted using TRIzol reagent (Mina Tajhiz Aria). The purity of total RNA was measured with the NanoDrop method (Thermo Fisher Scientific) at 260/280 nm. According to the manufacturer’s instructions, cDNA was synthesized using PrimeScript reverse transcriptase reagent kit (Takara Bio).

SYBR green real-time polymerase chain reaction
ACOX1 and NRF1 gene expression levels were evaluated using Step-One Plus Real-Time Instrument (ABI) with SYBR Premix Ex-TaqTM II kit (Takara). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a housekeeping gene (internal control). Primer sequences were designed with Primer Premier (Premier Biosoft International) for amplification of ACOX1, NRF1, and GAPDH (NM_001185039.2; NM_001185039; NM_001289745.1) (Table 1). Primer specificity was confirmed by Primer-BLAST (https://www.ncbi. nlm.nih.gov/tools/primer-blast). The master mix contained SYBR Premix Ex Taq II Bulk (SYBR green mix, RNase H, and dye from Takara Bio), forward and reverse primers (5 pmol), and template cDNA (see Table 1). The specificity of polymerase chain reactions was confirmed by analyzing the melting point curves of target and internal control genes.

Enzyme-linked immunosorbent assay
NRF1 and ACOX1 protein expression levels were evaluated using enzyme-linked immunosorbent assays (Bioassay Technology Laboratory). Results were measured with an enzyme-linked immunosorbent assay xs508M absorbance microplate reader (Biotek Instruments).

Statistical analyses
Statistical analysis was carried out using IBM SPSS Statistics for Windows, version 19. Data are presented as means ± SD. One-way analysis of variance was conducted to evaluate significant differences. P < .05 was considered statistically significant. The figures were formatted using GraphPad Prism 6.0 software.

Results

Descriptive characteristics
Among the 200 liver transplant recipients included in our study, 160 were men (age range, 49-54 years) and 40 were women (age range, 49-54 years). The HBV-positive group consisted of 120 men (81.07%; mean age 49 ± 10 years) and 30 women (20.27%; mean age 49 ± 10 years). The HCV-positive group consisted of 16 men (76.2%; mean age 49 ± 5 years) and 5 women (23.8%; mean age 49 ± 5 years). The HCC group consisted of 24 men (82.8%; mean age 54 ± 10 years) and 5 women (17.2%; mean age 54 ± 10 years). The ABO blood groups are summarized in Table 2.

Real-time polymerase chain reaction of ACOX1 and NRF1 gene expression
We found that NRF1 expression was increased in all 3 liver transplant recipient groups compared with the control group; however, this increase was not significant (P > .05; Figure 1). Furthermore, ACOX1 expression was decreased in all 3 liver transplant groups compared with the control group (P > .05; Figure 2).

Enzyme-linked immunosorbent assay
ACOX1 and NRF1 protein expression levels in all 3 liver transplant recipient groups were significantly decreased compared with the control group (P < .05; Figure 3).

Discussion

The present study investigated the association between ACOX1 and NRF1 gene and protein expression levels and presence of HBV, HCV, and HCC in liver transplant patients in South-Central Iran. Our results indicated that NRF1 and ACOX1 genes and their protein expression levels could be related to the development of CLD. Genetic changes have a significant role in viral hepatitis; therefore, identification of genes related to HCC and viral infections is essential.¹⁶

Treatment of CLDs can be assisted by the identification of genes in these diseases. So far, many genes have been identified and studied in connection with CLD. NRF1 is involved in many biological processes, such as protecting cholesterol homeostasis, adaptive responses to high cellular cholesterol, and hepatic toxicity.¹⁸ NRF1 plays a crucial role in the cellular adaptive antioxidant response regulation to oxidative stress processes.^18,20,29 In addition, NRF1 plays major roles in embryonic growth, organ differentiation, and hepatic lipid metabolism.¹⁹ Downregulation of NRF1 stimulates hepatocytes to oxidative stress-prompted cellular damage and toxicity.²² Previous studies have shown that NRF1 is overexpressed in many malignant tissues compared with normal tissues.³⁷ Interestingly, our results showed that NRF1 expression was increased in the 3 groups of liver transplant recipients (those with HCC, HBV, and HCV) compared with the control group; however, this increase was not significant. However, we did find that NRF1 protein expression in all 3 patient groups was significantly decreased compared with the control group (P < .05).

NRF1 overexpression may induce abnormal proliferation, which is also a hallmark of cancer cells.³⁸ However, there is no clear function of NRF1 in cancer cells. Niida and colleagues reported that the NRF1-binding motif associated with tumor malignancy might reflect hypermetabolism in aggressive tumors.³⁸ Amazingly, NRF1 expression in our HCC group was significantly increased by 2.25 (P < .05) compared with our HBV-positive group. Kim and colleagues reported that expression of NRF1 in tissues and cells is mainly determined at the mRNA level. In normal adult tissues, low levels of NRF1 transcript have been identified in the liver and pancreas.¹⁷ The mechanism for NRF1 mRNA and protein expression has not been fully understood in liver transplant patients, especially in those with viral hepatitis.¹⁶ At the same time, previous studies have indicated that disruption of NRF1 causes stress that starts ARE-driven genes in an NRF2-dependent manner.³⁹ NRF1 was also an antiapoptotic in developed hepatocytes.24 Hepatocyte-specific NRF1 knockout mice have been shown to develop hepatic steatosis.^26,29 Lee and colleagues showed that mice heterozygous for NRF1 presented with steatosis and liver endoplasmic reticulum stress.²⁸ Moreover, Koizumi and colleagues⁴⁰ showed that NRF1 is a short-lived protein that completely degraded the ubiquitin-proteasome pathway and posttranslational modification through accumulation, playing a role in stress responses.

ACOX1 is the prime and rate-preventive enzyme of the peroxisome β-oxidation pathway,^30,31 leading to H₂O₂ production.⁴¹ ACOX1 deregulation has been reported to induce peroxisomal disorders and carcinogenesis in the liver.⁴² In humans, ACOX1 deficiency is an autosomal recessive disorder that causes severe damage to nervous system function.⁴¹ Hematopoietic stem cell transplant has been used for ACOX1 deficiency (neonatal adrenoleukodystrophy). Hepatomegaly also occurs in patients with ACOX1 deficiency.⁴¹ Severe liver disease has been shown to develop in young ACOX1 knockout mice, characterized by steatosis, loss of peroxisomes, and disseminated hepatocyte death.⁴³ ACOX1 is the primary enzyme for DCA breakdown in humans and mice.³³ Abnormal urinary DCA was reported in ACOX1-deficient patients. Nevertheless, no information is available with regard to DCA in the liver of ACOX1-deficient patients and mice.³³

Activation of the peroxisome proliferator-activated receptor alpha (PPARα) was shown to be the cause of unfolded protein response in the endoplasmic reticulum of ACOX1^−/− liver samples.⁴ Our study showed that ACOX1 expression was decreased in all liver transplant groups compared with our control group (although not significantly, P > .05). However, we did find ACOX1 protein expression to be significantly decreased in all recipient groups (HCC, HBV, HCV) compared with the control group (P < .05). Our results agree with Zheng and colleagues,⁴² who reported that ACOX1 deficiency or decrease will cause activation of PPARα and induce endoplasmic reticulum stress, contributing to hepatocarcinogenesis (downregulation of ACOX1 upregulated PPARα expression).

Conclusions

Our results showed a possible association between ACOX1 and NRF1 gene expression and HBV and HCV virus infections and HCC in liver transplant patients (Shiraz, Iran). Results from this study suggested that ACOX1 and NRF1 could be new molecular markers in liver transplant and CLD. Further study is needed to explore the exact mechanism.

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