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ORIGINAL ARTICLE
Year : 2019  |  Volume : 32  |  Issue : 1  |  Page : 282-288

RECK gene promoter polymorphisms in patients with hepatocellular carcinoma along with chronic hepatitis C viral infection


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Hepatology, National Liver Institute, Menoufia University, Menoufia, Egypt
3 Department of Clinical Pathology, Ministry of Health, Bolak Al-Dakror General Hospital, Giza, Egypt

Date of Submission14-Nov-2017
Date of Acceptance31-Dec-2017
Date of Web Publication17-Apr-2019

Correspondence Address:
Heba A Mostafa
Birkit El-Saba, Menoufia 32651
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_763_17

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  Abstract 


Objective
The aim was to study possible associations of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene polymorphisms (rs11788747 and rs16932912) with the risk of developing hepatocellular carcinoma (HCC) among hepatitis C virus (HCV)-infected Egyptian patients.
Background
RECK is a membrane-anchored glycoprotein. Downregulation of RECK was found in a variety of neoplasms to be associated with poor survival and distant metastasis.
Patients and methods
The study included three groups: group I comprised 30 apparently healthy participants as a control group, group II comprised 35 patients with cirrhosis having HCV infection, and group III comprised 35 patients with cirrhosis having HCC along with HCV infection. PCR-restriction fragment length polymorphism was used for detection of RECK gene polymorphisms (rs11788747 and rs16932912).
Results
Regarding rs11788747, mutant AG/GG genotypes were statistically higher in HCC (60.0%) than in HCV and control groups (34.3 and 13.3%, respectively) and demonstrated 9.75 times (95% CI: 2.79–34.07) more risk of developing HCC compared with AA genotype (P < 0.001). G allele was statistically higher in HCC (51.4%) than HCV and control groups (22.9 and 8.3%, respectively). Regarding rs16932912, wild-type GG genotype was higher in HCC (71.4%) than in HCV and control groups (48.6 and 70.0%, respectively). G allele was statistically higher in HCC (81.4%) than HCV and control groups (71.4 and 83.3%, respectively) but without any statistical significance among groups regarding alleles.
Conclusion
RECK rs11788747 polymorphism could be involved in the pathogenesis of HCC, and rs16932912 polymorphism was not associated with the pathogenesis of HCC.

Keywords: chronic hepatitis C, genetic polymorphism, hepatocellular carcinoma


How to cite this article:
Abou-Elela DH, AlGhoraie AA, Mostafa HA. RECK gene promoter polymorphisms in patients with hepatocellular carcinoma along with chronic hepatitis C viral infection. Menoufia Med J 2019;32:282-8

How to cite this URL:
Abou-Elela DH, AlGhoraie AA, Mostafa HA. RECK gene promoter polymorphisms in patients with hepatocellular carcinoma along with chronic hepatitis C viral infection. Menoufia Med J [serial online] 2019 [cited 2019 May 21];32:282-8. Available from: http://www.mmj.eg.net/text.asp?2019/32/1/282/256143




  Introduction Top


Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the second most common cause of cancer-related mortality in eastern and western countries [1]. Each year, greater than 700 000 patients are diagnosed as having HCC, and morbidity rates of HCC continue to increase, possibly owing to deterioration of the environment, unhealthy dietary habits, and other related factors [2].

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), also known as RECK, is a membrane-anchored glycoprotein. This human gene is thought to be a metastasis suppressor against activated ras oncogenes [3]. The RECK gene is widely expressed in numerous normal tissues and non-neoplastic cell lines, but its expression is low or undetectable in oncogene-transformed fibroblasts or tumor-derived cell lines [4]. It is established that expression of RECK inhibits tumor invasion, metastasis, and angiogenesis in animal models. Moreover, studies illustrate that patients with high RECK expression in tumor tissues show better survival [5]. Thus, upregulation of RECK is considered as an expectant approach for treating malignant tumors [6].


  Aims Top


The aim was to study the role of RECK gene polymorphisms (rs11788747 and rs16932912) and the danger of developing HCC among hepatitis C virus (HCV)-infected Egyptian patients.


  Patients and Methods Top


The study included 100 participants divided into three groups: group I included 30 apparently healthy age-matched and sex-matched individuals as a control group (18 male and 12 female individuals), with mean age of 51.1 ± 5.7 years; group II included 35 patients having cirrhosis along with HCV (22 male and 13 female patients), with mean age of 53.7 ± 6.1 years; and group III included 35 patients with HCC (21 male and 14 female patients), with mean age of 54.7 ± 7.7 years. This study was carried out between March 2016 and August 2017 at Clinical Pathology Department, Faculty of Medicine, in collaboration with the National Liver Institute, Menoufia University. A written informed consent was provided by all participants, and an agreement to conduct the study was obtained from the ethics committee. All the participants were subjected to the following: full history taking, clinical examination, radiological investigations, and liver function tests (total and direct bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (GGT), alkaline phosphatase (ALP), international normalized ratio, total protein, and albumin). Hepatitis B surface antigen-positive patients and patients with secondary metastatic liver tumors were excluded from the study.

Sampling

Under complete aseptic conditions, 8 ml of venous blood was collected from each participant. Each blood sample was divided into three tubes: A, B, and C. Regarding tube A sample, 4 ml of blood was collected in a plain tube, left to clot at 37°C, and then centrifuged. Clear sera were separated and divided into three aliquots. The first aliquot was used to determine liver function tests such as total and direct bilirubin, AST, ALT, GGT, ALP, total protein, and albumin by Olympus AU640 chemistry analyzer (Diamond Diagnostic, Holliston, Massachusetts, USA). The second aliquot was used for measurement of α-fetoprotein (AFP) by enzyme-linked immunosorbent assay (ELISA) using Human AFP ELISA kits supplied by Glory Science Co. Ltd (Del Rio, Texas, USA). The third aliquot was used for detection of hepatitis markers (hepatitis B surface antigen and HCV antibody) by enzyme-linked immunosorbent assay (ELISA) using Human ELISA kits supplied by Adaltis (Freiburg, Germany). The specimens were kept frozen at −20°C until the time of assay.

Regarding tube B sample, 2 ml of blood was collected in a sterile sodium citrate vacutainer tubes for immediate assay of prothrombin time concentration and international normalized ratio. Regarding tube C sample: 2 ml of blood was collected in sterile vacutainer tubes containing EDTA for DNA extraction for evaluation of RECK gene polymorphisms.

DNA analysis

PCR-restriction fragment length polymorphism method was used to determine the distribution of genotype and allele frequencies of RECK single nucleotide gene polymorphisms (rs11788747 and rs16932912).

DNA extraction

The DNA was extracted using commercially available spin column technique (Scientific ZymoBead Genomic DNA Kits, Quick-gDNA MiniPrep) for DNA extraction from human whole blood (Zymo Research Corp, Freiburg, Germany). The eluted genomic DNA was stored in –80°C until amplification by PCR.

Primers

The lyophilized primers were supplied by Fermentas Life Sciences (Waltham, Massachusetts, USA). The lyophilized primers were reconstituted by addition of sterile water to a final concentration of 50 pmol/μl and distributed in aliquots and stored at −80°C.

For RECK rs11788747, forward primer was 5'-GTAGAAGAAGTGACTCATCC-3' and1the reverse primer was 5'-ATCTCACTCCGAAGA TAACC-3'.

For RECK rs16932912, forward primer was 5'-TGGAGATTGTTGATGCTC-3' and the reverse primer was 5'-CGGTACACAATGCTCAATAC-3'.

PCR amplification

PCR amplification was performed using MyTaq HS Red Mix master Mix Kit (Bioline USA Inc., Taunton, Massachusetts, USA), which is a ready-to-use 2 × mix for fast, highly specific, hot-start PCR. MyTaq HS Red Mix is powered by antibody-mediated hot-start and does not possess polymerase activity during the reaction setup, thus reducing nonspecific amplification. The following protocol is for 25 ml reaction: template 2 ml, primers 1 ml, MyTaq HS Red Mix 2 × 12.5 ml, and water (dH2O) up to 25 ml.

PCR cycling conditions

One cycle of initial denaturation at 95°C for 1 min, then 25–35 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and extension at 72°C for 10 s. After the last cycle, a final extension of 4 min at 72°C was done. The amplification products were separated by electrophoresis on 2% agarose gel stained with ethidium bromide and visualized on an ultraviolet transilluminator. PCR products were subjected to digestion by restriction enzyme RsaI (New England Bio Labs Inc., Ipswich, Massachusetts, USA) for rs11788747 and Tfil (New England Bio Labs Inc.) for rs16932912. The amplification products were separated by electrophoresis on 2% agarose gel stained with ethidium bromide and visualized on an ultraviolet transilluminator. The products in RECK rs16932912 were G/G at 353bp and A/A at 250 and 103bp, and in RECK rs11788747 were A/A at 242bp and G/G at 140 and 102bp.

Statistical analysis

The data were collected, formulated, and statistically analyzed using SPSS version 17 (SPSS Inc., Chicago, Illinois, USA). Descriptive statistics were in the form of mean ± SD for parametric data. χ2-Test is used to compare between two groups or more regarding one qualitative variable. One-way analysis of variance test was used for comparison between three or more groups having quantitative variables normally distributed. Kruskal–Wallis test was used for comparison between three or more groups not normally distributed. Odds ratio and CI were calculated by logistic regression analysis. Mann–Whitney U-test is a nonparametric test of significance used for comparison between two groups not normally distributed having quantitative variables. P value showed significant difference if P less than 0.05, nonsignificant difference if P greater than 0.05, and highly significant difference if P less than 0.001.


  Results Top


The study included 100 participants divided into the three groups: group I comprised 30 apparently healthy age-matched and sex-matched participants as a control group (18 male and 12 female individuals) and their mean age was 51.1 ± 5.7 years; group II comprised 35 patients with cirrhosis having HCV (22 male and 13 female patients), and their mean age was 53.7 ± 6.1 years; and group III comprised 35 patients with HCC (21 male and 14 female patients), and their mean age was 54.7 ± 7.7 years.

There was a statistically high significant difference between groups regarding laboratory finding that included AST, ALT, GGT, albumin, direct bilirubin, pothrombin concentration, and AFP (P < 0.001) [Figure 1].
Figure 1: Laboratory findings of studied groups.

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The genotype dispersion of RECK rs11788747 single nucleotide polymorphism showed that AA wild-type genotype was higher in control group (86.7%) compared with HCV and HCC groups (65.7 and 40.0%, respectively), and then, AG/GG mutant genotype was higher in HCC groups (60.0%) than in HCV and control groups (34.3 and 13.3%, respectively). No statistical distinction was found between AG/GG genotypes and AA among control and HCV groups (P = 0.08%), whereas there was a statistically significant difference among control and HCC groups (P < 0.001%); moreover, there was a statistically significant difference among HCV and HCC groups (P = 0.03). Regarding alleles frequencies, A allele was more expressed in control group (91.7%) than in HCV and HCC groups (77.1 and 48.6%, respectively), whereas G allele was higher in HCC group (51.4%) than in HCV and control groups (22.9 and 8.3%, respectively) [Table 1].
Table 1: Reversion-inducing cysteine-rich protein with Kazal motifs genotypic frequencies (11788747 and 16932912) and allele polymorphism among studied groups

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As to genotype distribution of RECK rs16932912 single nucleotide polymorphism, there was no statistically significant difference between wild-type genotype GG and AA/AG mutant genotypes among groups (P = 0.09). As to alleles frequencies, A allele was more expressed in HCV (28.6%) than in HCC and control groups (18.6 and 6.7%, respectively), whereas G allele had similar frequency in HCC, control, and HCV groups (81.4, 83.3, and 71.4%, respectively), demonstrating no significant difference in distribution of the two alleles (G and A) among the three groups (P = 0.19) [Table 1].

In the present study, the risk of RECK rs11788747 SNP, regarding AG/GG genotypes, among HCC cases versus control and HCV groups (9.75 and 2.88, respectively) times than wild-type AA genotype and are more risky for HCC (CI: 2.79–34.07 and 1.09–7.60, respectively). However, in RECK rs16932912 SNP, odds ratio cleared that neither AA/AG nor GG genotype carries the risk of HCC [Table 2].
Table 2: Risk of reversion-inducing cysteine-rich protein with Kazal motifs gene polymorphism (11788747 and 16932912) among hepatocellular carcinoma cases versus controls and hepatocellular carcinoma cases versus hepatitis C virus cases

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The present study also found in HCC group that regarding RECK gene rs11788747 SNP, lymph node metastasis was statically significant higher in AG/GG mutant genotypes than wild-type AA genotype (P < 0.001) [Table 3]. Distant metastasis also was statistically higher in AG/GG mutant genotypes than wild-type AA genotype (P = 0.02) [Table 3]. However, in RECK rs16932912 SNP, there was no statically significant difference between wild-type genotype GG and AA/AG mutant genotypes in HCC group with distant and lymph node metastasis [Table 3].
Table 3: Reversion-inducing cysteine-rich protein with Kazal motifs genotypic frequencies in hepatocellular carcinoma cases regarding lymph node metastasis and distant metastasis

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Comparison between different rs11788747 and rs1693291 gene polymorphisms among group II and among group III regarding laboratory findings revealed no statistically significant difference [Table 4].
Table 4: Comparison between different rs11788747and rs1693291 gene polymorphism among group II and among group III regarding laboratory findings

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  Discussion Top


HCC is the fifth most common malignancy in men worldwide and the second most incessant reason for malignancy-related deaths [7]. Hepatocarcinogenesis is a multi-step process where dysplastic macronodules in cirrhosis modify bit by bit into HCC [8]. Numerous markers are accessible for the determination of HCC; however, none of them have adequate sensitivity and specificity. For example, AFP, the commonest utilized serum marker for the analysis of HCC clinically has sensitivity and specificity of 41–65% and 80–94%, respectively [9].

A RECK mRNA expression is considered as a tumor suppressor gene in HCC tumor tissues in patients with better survival and less invasive characteristics [10]. RECK is considered as a tumor suppressor gene that contrarily regulates matrix metalloproteinases and communicates in various normal human tissues, yet it is downregulated in some human tumors and has been decidedly connected with the survival of patients with cancer [11],[12].

The RECK gene structure has been located on the chromosome region 9p13→p12. The RECK gene incorporates 21 exons and 20 introns, and 13 SNPs have been recognized. Among 13 SNPs, rs16932912 and rs11788747 were found inside the coding succession in exons 9 and 13, respectively [5].

Gene promoter hypermethylation has been connected with the silencing of tumor-related genes, which is viewed as the most critical epigenetic disturbance in numerous tumors [13].

Zhang et al. [14] recommended that hypermethylation may prompt promoter silencing of RECK mRNA, related with a low survival rate in HCC. Moreover, they found that RECK mRNA was downregulated in HCC tissues than in non-HCC tissues. Expression of RECK was mostly diminished in patients with HCC with hypermethylation than those with demethylation, with significant relationship between RECK mRNA and poor survival after surgery.

This study was planned to examine the effects of single nucleotide polymorphisms of RECK (rs16932912 and rs11788747) on HCC susceptibility in three groups, a group of healthy individuals, HCV-related cirrhosis group, and HCC along with HCV infection group, looking for a possible association that might exist with the risk of developing HCC.

The genotype dispersion of RECK rs11788747 SNP was as follows: AA genotype was higher in control group comparing with HCV and HCC groups, and AG/GG genotype was higher in HCC groups than in HCV and control groups. This result was in accordance with Bahgat et al. [15], who found that RECK rs11788747 A/G and G/G genotype frequencies were higher in patients with HCC compared with the controls.

Regarding allele frequencies, A allele was more expressed in control group than in HCV and HCC groups, whereas G allele was higher in HCC group than in HCV and control groups. Thus, in the present study, the presence of at least one polymorphic G allele increases the susceptibility to HCC compared with the A/A wild-type carriers. This in concurrence with Bahgať et al. [15] who found that RECK rs11788747 A/G and G/G genotypes frequencies were significantly higher in patients with HCC compared with the healthy controls.

As to genotype distribution of RECK rs16932912 SNP, no statistically significant difference between the rs16932912 wild-type genotype GG and mutant genotypes AG/AA was found. This finding was in concurrence with that studies by Chung et al. [5] and Badawy et al. [16] that proposed RECK gene rs16932912 polymorphism was not a hazard factor in expanding HCC susceptibility and distant metastasis.

As to alleles frequencies, A allele was more expressed in HCV than in HCC and control groups, whereas G allele was higher in HCC group than in control and HCV groups, demonstrating no significant difference in the distribution of the two alleles (G and A) among the three groups.

This study also revealed that there was no huge relationship between the RECK genotype rs11788747 polymorphisms and laboratory results. This agreed with Chung et al. [5] who studied the effect of other RECK gene SNPs on ALT, AST, and AFP. They found no significant difference for all results, with the exception of ALT levels, which were significantly different between the rs11788747 AA and AG/GG genotypes. However, the study by Gaber et al. [17], evaluated RECK rs11788747 genotypes and its connection to laboratory results. Mutant genotypes AG/GG frequencies indicated elevated levels of ALT, AST, and ALP versus wild-type AA genotype. Additionally, Bahgat et al. [15] conducted a study among the three distinctive genotype groups with respect to the laboratory results: AST, ALT, and AFP. There was a significant difference among the three groups regarding these laboratory data, except for AST.


  Conclusion Top


RECK gene polymorphism (rs11788747) could be implicated in the pathogenesis of HCC, and presence of A/G genotype in rs16932912 was not involved in the development of HCC.

Further independent studies using a larger population, studying genotypic phenotypic associations, and including both sexes are necessary to clarify the relation between RECK gene polymorphism (rs11788747) and pathogenesis of HCC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Zhu J, Ling Y, Xu Y, Lu M, Liu Y, Zhang C. Promoter hypermethylation of the RECK gene is associated with its low expression and poor survival of esophageal squamous cell carcinoma. Oncol Lett 2017; 3:1911–1918.  Back to cited text no. 12
    
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