|Year : 2017 | Volume
| Issue : 2 | Page : 626-632
Matrix metalloproteinase-1 polymorphism in hepatocellular carcinoma patients with hepatitis C or B
Gehan K El Saeed1, Belal A. E. Montaser1, Abdel M Aoda2, Omnia S. N. Ahmed3
1 Clinical Pathology Department, Faculty of Medicine, Menoufia University, Shebein El Kom, Egypt
2 Hepatology Department, National Liver Institute, Menoufia University, Shebein El Kom, Egypt
3 Clinical Pathology Department, National Liver Institute, Menoufia University, Shebein El Kom, Egypt
|Date of Submission||10-Jan-2017|
|Date of Acceptance||16-Apr-2017|
|Date of Web Publication||25-Sep-2017|
Omnia S. N. Ahmed
Clinical Pathology Department, National Liver Institute, Menoufia University, Shebein El Kom, Menoufia Governorate, 32511
Source of Support: None, Conflict of Interest: None
The aim of this work was to study the relations hip between matrix metalloproteinase-1 (MMP-1) −1607 1G/2G gene polymorphism and hepatocellular carcinoma (HCC) in patients infected with hepatitis B or C viruses.
MMP-1, an interstitial collagenase, plays an important role in the breakdown of extracellular matrix. It has been demonstrated that the overexpression of this enzyme is associated with tumor initiation, invasion, and metastasis. The − 1607 single guanine (1G)-to-2G polymorphism (reference single-nucleotide polymorphism 1799750) in the MMP-1 promoter region creates an E26 (Ets)-binding site and results in transcriptional upregulation. Therefore, the MMP-1 polymorphism may influence an individual's susceptibility to the development of certain tumors such as HCC.
Patients and methods
This study was carried out on 100 participants; 40 of them were patients with HCC infected with hepatitis C virus, 20 of them were patients with HCC infected with hepatitis B virus, and the remaining 40 were age-matched and sex-matched healthy volunteers. All participants were subjected to full history taking, laboratory investigations including liver profile, α-fetoprotein, and prothrombin induced by vitamin K absence II, and determination of MMP-1 − 1607 1G/2G polymorphism by PCR–restriction fragment length polymorphism assay.
The MMP-1 − 1607 genotype distribution among HCC patients was significantly different from that in healthy controls (P < 0.001). Compared with the wild-type 1G/1G genotype, the variant 1G/2G and 2G/2G genotypes and the 2G allele were associated with risk for HCC (P < 0.001). The 1G/2G genotype was associated with large tumor size. The 1G/2G and 2G/2G genotypes were associated with Child–Pugh B and C. The 1G/2G genotype was associated with advanced tumor stage and the 2G/2G genotype was associated with metastatic tumor stage.
The results suggest that the MMP-1 − 1607 1G/2G polymorphism is associated with an increased risk for HCC.
Keywords: hepatocellular carcinoma, matrix metalloproteinase-1, polymerase chain reaction-restriction fragment length polymorphism, polymorphism
|How to cite this article:|
El Saeed GK, Montaser BA, Aoda AM, Ahmed OS. Matrix metalloproteinase-1 polymorphism in hepatocellular carcinoma patients with hepatitis C or B. Menoufia Med J 2017;30:626-32
|How to cite this URL:|
El Saeed GK, Montaser BA, Aoda AM, Ahmed OS. Matrix metalloproteinase-1 polymorphism in hepatocellular carcinoma patients with hepatitis C or B. Menoufia Med J [serial online] 2017 [cited 2019 Jun 17];30:626-32. Available from: http://www.mmj.eg.net/text.asp?2017/30/2/626/215477
| Introduction|| |
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and its incidence has increased worldwide in recent decades, making it currently the fifth most common form of malignancy in men and the ninth in women, with a male/female ratio of 2.4. Every year, there are ~700 000–1 000 000 new cases and about 600 000–800 000 of them die from the disease, making HCC the second leading cause of death by cancer in the world .
The main etiology of HCC is liver cirrhosis caused by chronic hepatitis B virus (HBV) or hepatitis C virus (HCV), alcohol consumption, fatty liver diseases, or, less commonly, by autoimmune or genetic or metabolic liver diseases .
Matrix metalloproteinase-1 (MMP-1), a member of the MMP family, is an endogenous peptide enzyme that is most widely expressed in interstitial collagenase, which can degrade the extracellular matrix. It is involved in many stages of tumorigenesis, such as in promotion of tumor growth through stimulation of cellular proliferation, invasion, and migration, in angiogenesis, and in suppression of tumor cell apoptosis .
MMP-1 − 1607 1G/2G (rs1799750) contains a guanine insertion/deletion polymorphism at position − 1607 and is a functional single-nucleotide polymorphism (SNP) that can upregulate MMP expression. The association between the MMP-1 − 1607 1G/2G polymorphism and the emergence and development of several diseases including the risk for many cancers has been reported .
Individuals with the 2G/2G genotype of MMP-1 − 1607 1G/2G polymorphism were reported to have higher MMP-1 level. Highly expressed MMP-1 played a remarkable role in degrading collagens I and III, which was very important to cancer metastasis. In a sense, it heralded poorer prognosis in malignant patients .
| Patients and Methods|| |
This study was conducted at the Clinical Pathology Department, Faculty of Medicine, Menoufia University, during the period from March 2015 to November 2016.
This study was carried out on 100 participants; 40 of them were HCC patients infected with HCV, 20 of them were HCC patients infected with HBV, and the remaining 40 were healthy volunteers. Approval of the ethical committee and patient consent were obtained. The control group included individuals who had no proven malignant disease, no history of liver disease, and no serological evidence of HBV or HCV infection. The diagnosis of HCC cases included a combination of history, clinical examination, radiological examination including ultrasound and triphasic computed tomography, Child–Pugh classification, and laboratory investigations including hepatitis C and B markers, prothrombin induced by vitamin K absence II (PIVKA II), and α-fetoprotein (AFP). The samples were collected before chemotherapeutic or radiation therapy treatment.
All participants were subjected to full history taking, clinical examination, abdominal ultrasound, and laboratory tests including complete blood count (Sysmex XT 1800; Sysmex America, Inc. One Nelson C. White Pkwy, Mundelein, USA), liver function tests (Roch Integra 400 plus; Roche Diagnostics Ltd.CH-6343, Rotkreuz, Switzerland), AFP (Hitachi Cobas E 411; Roche Diagnostics Ltd CH-6343, Rotkreuz. Switzerland), HCVAb and HBsAg (Hitachi Cobas E 411; Switzerland), PIVKA II level by enzyme-linked immunosorbent assay, and determination of MMP-1 gene polymorphisms by PCR–restriction fragment length polymorphism (RFLP).
DNA extraction and genotyping
Genomic DNA was isolated from ethylenediaminetetraacetic acid-preserved whole blood by standard proteinase K digestion ethanol precipitation using the Thermo Scientific Gene JET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). After ethanol precipitation, the DNA was purified and dissolved in double-distilled water and frozen at −20°C until use. The MMP-1 − 1607/2G genotype was determined by PCR-RFLP assay. The PCR primers (Thermo Fisher Scientific) were as follows: forward 5′-TCG TGA GAA TGT CTT CCC ATT-3′ and reverse 5′-TCT TGG ATT GAT TTG AGA TAA GTG AAA TC-3′. PCR cycling conditions were 1 min at 95°C, followed by 35 cycles of 1 min at 95°C, 35 s at 55°C, and 35 s at 72°C, with a final elongation step at 72°C for 5 min. For RFLP, the PCR products were digested with 5 U of Xmn I enzyme (New England Biolabs, Ipswich, MA, USA) at 37°C and visualized by electrophoresis on 3% agarose under ultraviolet transillumination. The allele types were determined as follows: a single 117 bp fragment for the 2G/2G genotype, two fragments of 28 and 89 bp for the 1G/1G genotype, and three fragments of 28, 89, and 117 bp for the 1G/2G genotype .
Statistical analysis of this study was conducted using SPSS version 20.0 (SPSS Inc., Chicago, Illinois, USA). Data were expressed as descriptive and analytical. The χ2-Test, t- test, Mann–Whitney U-test, Kruskal–Wallis test, F-test, post-hoc test, Fisher's exact test, odds ratio (OR), and 95% confidence interval (CI) tests were used. P values greater than 0.05 were considered statistically nonsignificant, those less than 0.05 were considered statistically significant, and values less than 0.001 were considered statistically highly significant.
| Results|| |
Baseline characteristics of the patients and controls are as follows: There were no significant differences between the two groups in terms of age and sex [Table 1] but there were statistically significant differences regarding alanine aminotransferase, aspartate aminotransferase, total and direct bilirubin, serum albumin, AFP, PIVKA II, and international normalized ratio (INR) [Table 2].
The frequency distributions of the different genotypes for MMP-1 − 1607 1G/2Gpolymorphism are shown in [Table 3]. There was a significantly higher proportion of individuals with the 1G/1G genotype in the control group than in the patient group (85 vs. 8.3%, P < 0.001), a significantly lower proportion of individuals with the 2G/2G genotype in the control group than in the patient group (5 vs. 35%, P < 0.001), and a significantly lower proportion of individuals with the 1G/2G genotype in the control group than in the patient group (10 vs. 56.7%, P < 0.001). There was a highly significantly lower prevalence of the 2G allele in the control group than in the patient group (10 vs. 63.3%, P < 0.001) [Table 3].
On evaluating the risk for HCC according to the MMP-1 − 1607 genotype using the 1G/1G genotype as the reference, we found that the 1G/2G genotype was highly associated with increased risk for HCC (OR = 57.80, 95% CI = 14.28–233.95, P < 0.001); further, the 2G/2G genotype was associated with increased risk for HCC (OR = 71.40, 95% CI = 12.69–401.83, P < 0.001) [Figure 1] and [Figure 2].
|Figure 1: Representative agarose gel electrophoresis of matrix metalloproteinase-1 gene amplification bands that correspond to ladder band size of 117 bp (lanes 2 and 3) and 50 bp (lane 1) DNA ladder.|
Click here to view
|Figure 2: Representative agarose gel electrophoresis of PCR–restriction fragment length polymorphism analysis of matrix metalloproteinase-1 - 1607 rs1799750 genotypes in genomic DNA of participants with restriction enzyme XmnI. Lane 1, 50 bp DNA ladder; lanes 2 and 4 (1G/1G), band 89 and 28 bp; lanes 6 and 8 (1G/2G), band 117, 89, and 28 bp; lanes 3, 5, 7, 9, and 10 (2G/2G), band 117 bp.|
Click here to view
Compared with the 1G allele, it was found that the 2G allele was highly associated with increased risk for HCC (OR = 15.55, 95% CI = 6.85–35.27, P < 0.001) [Table 3].
To assess the role of the MMP-1 − 1607gene polymorphism in the clinicopathological features of HCC cases, the distribution of clinical and pathological parameters among MMP-1 − 1607 genotypes was estimated. There was a statistically significant difference between 1G/2G and 1G/1G genotypes with respect to the size of the focal lesion: the study found a significantly higher prevalence of the 1G/2G genotype compared with the 1G/1G in large focal lesions (82.4 vs. 40%) and a significantly lower prevalence of the 1G/2G genotype compared with the 1G/1G genotype in small and medium focal lesions. There was a statistically significant difference between the three genotypes with regard to Child–Pugh classification: there was a higher prevalence of 2G/2G and 1G/2G genotypes compared with the 1G/1G genotype in individuals with Child B and C, and a lower prevalence of 2G/2G and 1G/2G genotypes than of the 1G/1G genotype in individuals with Child A. There was a significant difference between the three genotypes regarding stage of HCC: there was a higher prevalence of the 1G/2G genotype compared with the 2G/2G and 1G/1G genotypes in advanced-stage tumor (P < 0.001), a higher prevalence of the 2G/2G genotype than of the 1G/2G and 1G/1G genotypes in metastatic-stage tumor (P < 0.001), and a lower prevalence of the 1G/2G and 2G/2G genotypes than of the 1G/1G genotype in early-stage tumor (P < 0.001) [Table 4].
|Table 4: Tumor characteristics of the studied genotype groups among total patients|
Click here to view
On using the 1G/1G genotype as the reference genotype, 1G/2G and 2G/2G genotypes were found to be associated with a low level of albumin and prothrombin concentration and a high level of INR; in addition, the 2G/2G genotype was associated with a high level of total bilirubin when compared with other genotypes [Table 5].
|Table 5: Distribution of the laboratory investigations according to the studied genotype in patients|
Click here to view
| Discussion|| |
HCC is a global problem and its epidemiological data vary across regions. HCC is one of the health problems facing the health authorities in Egypt. Studies report an almost two-fold increase in HCC among chronic liver disease patients over a decade .
Liver carcinogenesis is a complex and multifactorial process in which both environmental and genetic features interfere and contribute to malignant transformation .
Numerous candidate gene studies have reported associations between SNPs and the presence of HCC, whereas limited GWAS studies have shed light into additional unsuspected loci that might be involved in hepatic carcinogenesis .
MMP-1 is a secreted protein belonging to the collagenase group. Increased MMP-1 expression is observed in various conditions, including inflammation, wound healing, chronic degenerative disease, and cancers. MMP-1 promotes tumor progression not only through extracellular matrix (ECM) degradation of native fibrillar types I–III and V collagens but also through regulation of the function of biologically active molecules by releasing them from ECM stores .
The − 1607 single guanine (1G)-to-2G polymorphism (reference SNP 1799750) in the MMP-1 promoter region creates an E26 (Ets)-binding site and results in transcriptional upregulation .
This work aimed to study the MMP-1 gene polymorphism in patients with HCC infected with HCV or HBV and its relation to the disease characteristics.
In this study, the mean age of the patients with HCC was 54.76 ± 5.47 years, a finding that may indicate that HCC is more commonly encountered in old ages. These results agreed with Luo et al. , who found that the mean age of HCC patients was 54.1 ± 10.5 years, and with the findings of Shaker et al. , who found that the most frequent age category affected by HCC was 51–60 years. On the other hand, higher age incidence was reported by Giakoumidakis et al. , who found that HCC was more frequent in patients 65 years or older.
In this study, 68.3% of HCC patients were male and 31.7% were female. This was in agreement with the results of Rizk et al. , who found that most patients with HCC were male, and with the findings of Abdelgawad et al. , who reported that 80% of patients with HCC were male and 20% were female.
De Lope et al. suggested that this male predominance may be due to higher rates of exposure to liver carcinogens and hepatitis virus infections in men or to an estrogen-mediated inhibition of interleukin-6 production by Kupffer cells in the female population, leading to reduced liver injury and compensatory proliferation.
Considering the MMP-1 − 1607 1G/2G polymorphism, this study showed statistically significant difference in the distribution of the genotypes and allele frequencies between HCC patients and healthy controls. The results showed a significantly higher prevalence of the 1G/1G genotype in the control group than in the patient group (85 vs. 8.3%), a significantly lower prevalence of the 1G/2G genotype in the control group than in the patient group (10 vs. 56.7%), and a significantly lower prevalence of the 2G/2G genotype in the control group than in the patient group (5 vs. 35%). The study showed a significantly higher prevalence of the 1G allele in the control group than in the patient group (90 vs. 36.7%) and a significantly lower prevalence of the 2G allele in the control group than in the patient group (10 vs. 63.3%) [Table 3].
The results showed that the 1G/2G and 2G/2G genotypes were highly associated with increased risk for HCC (P < 0.001) when compared with the 1G/1G genotype. Compared with the 1G allele, it was found that the 2G allele was highly associated with increased risk for HCC (P < 0.001) [Table 3].
Similarly, Okamoto et al.  found that the number of 2G/2G homozygotes of the MMP-1 − 1607 1G/2G polymorphism increased in HCC patients.
Our results are similar to those obtained by Han et al. , who stated that MMP-1 rs1799750 was associated with increased risk for cancers such as lung, colorectal, head, and neck.
The obtained data were similar to those of Zang et al. , who found that MMP-1 − 1607 1G/2G and 2G/2G variants presented more frequently in osteosarcoma patients.
In contrast with the present study, Kazimi et al.  stated that genotypic distributions of MMP-1 − 1607 1G/2G polymorphisms were in accordance with Hardy–Weinberg equilibrium in HCC patients and in the control group (P > 0.05). In the case–control analysis, the distribution of genotypes and allele frequencies did not differ from those in the control group (P > 0.05).
In HBV patients with or without HCC, the genotype distribution of the MMP-1 − 1607 polymorphism was similar ,.
This study showed that there was a statistically significant relationship between the 1G/2G genotype and large tumor size when compared with the 1G/1G genotype. Genotypes 1G/2G and 2G/2G were significantly related to Child B and C when compared with the 1G/1G genotype. There was a high statistically significant relationship between the 1G/2G genotype and advanced tumor invading the portal vein, and between the 2G/2G genotype and metastatic tumors [Table 4].
The present study found that 1G/2G and 2G/2G genotypes were associated with a low level of albumin and prothrombin concentration and a high level of INR when compared with the 1G/1G genotype. In addition, the 2G/2G genotype was associated with a high level of total bilirubin when compared with other genotypes [Table 5].
Okamoto et al.  explained that the higher transcriptional activity of the 2G allele caused MMP-1 overexpression in host tissues, which increased the invasive ability of tumor cells.
Kazimi et al.  found that MMP genotypes were not significantly associated with clinicopathological parameters like tumor histological differentiation grade, dimension number of the tumor, and portal vein invasion, except for the MMP-1 − 1607 1G/2G polymorphism 2G/2G genotype, which was associated with portal vein invasion (P < 0.02).
In contrast with the present study, Hettiaratchi et al.  reported that 2G homozygosity was associated with a favorable prognosis for colorectal carcinoma patients.
Okazaki et al.  reported that the MMP-1 protein and mRNA were associated with growth of the small-sized tumors in which cancer cells invade the portal tract and fibrous bands. Taken together, the role of the MMP-1 − 1607 1G/2G gene polymorphism in cancer progression is still controversial.
If the present findings are confirmed in both larger series and other ethnic origins, genetic testing of the MMP-1 − 1607 1G/2G polymorphism may be useful in detecting high-risk individuals such as hepatitis virus seropositive individuals who are at an even greater risk for HCC. The results may encourage the higher-risk population to have frequent medical examinations to detect early-stage HCC. Furthermore, the knowledge of the mechanisms involved in HCC carcinogenesis may help identify targets for the development of chemoprevention or therapeutic strategies.
In conclusion, the current results indicate that the 1G-to-2G polymorphism in MMP-1 is a genetic susceptibility factor for both development and progression of HCC. Both 1G/2G and 2G/2G genotypes and the 2G allele are associated with increased risk for HCC. The 1G/2G genotype significantly increased in large-sized tumor. The 1G/2G and 2G/2G genotypes significantly increased in patients with Child B and C. There was a high statistically significant relationship between the genotype 1G/2G and advanced tumor and between the 2G/2G genotype and metastatic tumors.
The obtained results were consistent with the biologic function of the polymorphism and supported the hypothesis that aberrant MMP-1 expression may play a pivotal role in cancer development and progression. However, the molecular mechanism and signal pathways associated with the MMP-1 − 1607 1G/2G polymorphism in HCC still needs further study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Lopes FLM, Coelho FF, Kruger JAP, Fonseca GM, Araujo RLC, Jeismann VB, Herman P
Influence of hepatocellular carcinoma etiology in the survival after resection. Arq Bras Cir Dig 2016; 29:105–108.
Yim H, Suh S, Um S. Current management of hepatocellular carcinoma: An Eastern perspective. World J Gastroenterol 2015; 21:3826–3842.
Efendioglu M, Basaran R, Bayrak OF, Isik N, Kaner T, Sahin F, et al.
The role of a single nucleotide polymorphism of the matrix metalloproteinase-1 gene promoter region in invasion and prognosis of meningiomas. Turk Neurosurg 2014; 24:731–736.
Zhang Y, Pang Z, Li H. Association of matrix metalloproteinase-1 polymorphism with the risk and prognosis of osteosarcoma patients. Int J Clin Exp Pathol 2016; 9:10430–10436.
Wu D, Song P, Fu B, Lu M, Zhao Q, Wang B. Genetic polymorphisms of matrix metalloproteinase-1 is associated with the risk of cancer metastasis: An update meta-analysis. Int J Clin Exp Med 2016; 9:1435–1446.
Okamoto K, Ishida C, Ikebuchi Y, Mandai M, Mimura K, Murawaki Y, et al.
The genotypes of IL-1 beta and MMP-3 are associated with the prognosis of HCV-related hepatocellular carcinoma. Inter Med 2010; 49:887–895.
Shaker M. Epidemiology of HCC in Egypt. J Gastroenterol Hepatol 2016; 4:00097.
Nahon P, Zucman-Rossi J. Single nucleotide polymorphisms and risk of hepatocellular carcinoma in cirrhosis. J Hepatol 2012; 57:663–674.
Naik A, Košir R, Rozman D. Genomic aspects of NAFLD pathogenesis. Genomics 2013; 102:84–95.
Chen YK, Tung CW, Lee JY, Hung YC, Lee CH, Chou SH, et al.
Plasma matrix metalloproteinase 1 improves the detection and survival prediction of esophageal squamous cell carcinoma. Sci Rep 2016; 6:30057.
Liu L, Wu J, Wu C, Wang Y, Zhong R, Zhang X, et al.
functional polymorphism (−1607 1G → 2G) in the matrix metalloproteinase-1 promoter is associated with development and progression of lung cancer. Cancer 2011; 117:5172–5181.
Luo J, Zhang Z, Liu Q, Zhang W, Wang J, Yan Z. Endovascular brachytherapy combined with stent placement and TACE for treatment of HCC with main portal vein tumor thrombus. Hepatol Int 2015; 10:185–195.
Shaker MK, Abdella HM, Khalifa MO, El Dorry AK. Epidemiological characteristics of hepatocellular carcinoma in Egypt: A retrospective analysis of 1313 cases. Liver Int 2013; 33:1601–1606.
Giakoumidakis K, Nikolaos F, Ioannis E, Evangelos A, Vasileios P, Hero B. Frequency and predisposing factors of hepatocellular carcinoma in a Hepatology Outpatient Unit. Health Sci J 2015; 9:1–10.
Rizk E, Zakaria S, Abdel-Razik A, Farouk N. Soluble CD25 and hepatocellular carcinoma. Int J Adv Res 2015; 3:658–664.
Abdelgawad IA, Mossallam GI, Radwan NH, El-zawahry H, El-hefnawy M. Can glypican3 be diagnostic for early hepatocellular carcinoma among Egyptian patients? Asian Pac J Cancer Prev 2013; 14:7345–7349.
De Lope C, Tremosini S, Forner A, Reig M, Bruix J. Management of HCC. J Hepatol 2012; 56:75–87.
Han G, Wei Z, Lu Z, Cui H, Bai X, Ge H, et al.
Association between matrix metalloproteinase 1 − 1607 1G>2G polymorphism and cancer risk: A meta-analysis including 19706 subjects. Int J Clin Exp Med 2014; 7:2992-2999.
Kazimi MC, Nalbantoglu S, Kiliç M, Berdeli A. Clinical and genetic aspects of Turkish hepatocellular carcinoma patients: Results of a single center study. Int J Phys Sci 2010; 5:2379–2392.
Zhai Y, Qiu W, Dong XJ, Zhang XM, Xie WM, Zhang HX, et al.
Functional polymorphisms in the promoters of MMP-1, MMP-2, MMP-3, MMP-9, MMP-12 and MMP-13 are not associated with hepatocellular carcinoma risk. Gut 2007; 56:445–447.
Langers AMJ, Verspaget HW, Hommes DW, Sier CFM. Single nucleotide polymorphisms of matrix metalloproteinases and their inhibitors in gastrointestinal cancer. World J Gastrointest Oncol 2011; 3:79–98.
Hettiaratchi A, Hawkins NJ, McKenzie G, Ward RL, Hunt JE, Wakefield D, et al.
The collagenase-1 (MMP-1) gene promoter polymorphism − 1607/2G is associated with favourable prognosis in patients with colorectal cancer. Br J Cancer 2007; 96:783–792.
Okazaki I, Wada N, Nakano M, Saito A, Takasaki K, Doi M, et al.
Difference in gene expression for matrix metalloproteinase-1 between early and advanced hepatocellular carcinomas. Hepatology 1997; 25:580–584.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]