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  Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 52  |  Issue : 2  |  Page : 251-254
 

Heterozygosity of stromelysin-1 (rs3025058) promoter polymorphism is associated with gastric cancer


1 Department of Cell Biology, Institute of Genetics and Hospital for Genetic Diseases, Hyderabad, India
2 Department of Gastroenterology, Gandhi Hospital, Secunderabad, Andhra Pradesh, India
3 Department of Genetics, Osmania University, Hyderabad, India

Date of Web Publication5-Feb-2016

Correspondence Address:
A Venkateshwari
Department of Cell Biology, Institute of Genetics and Hospital for Genetic Diseases, Hyderabad
India
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Source of Support: Financial support from University Grants Commission (Research Fellowship in Science for Meritorious Students to Krishnaveni D) is kindly acknowledged, Conflict of Interest: None


DOI: 10.4103/0019-509X.175806

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 » Abstract 

Background: Gastric cancer (GC) is the third most common cancer in India and is mediated by multiple genetic, epigenetic and environmental risk factors. A single nucleotide polymorphism rs3025058 at − 1171 of the stromelysin-1 (matrix metalloproteinase [MMP]-3) promoter is resulting due to insertion/deletion of adenine thought to have an impact on increasing the risk for tumor formation. Aim: This study is aimed to understand the role of stromelysin-1 rs3025058 (−1171, 5A/6A) promoter polymorphism in the etiology of GC in Indian population. Materials And Methods: Genomic DNA was isolated from blood samples of the GC patients and controls. The genotyping of stromelysin-1 rs3025058 (−1171, 5A/6A) promoter polymorphism was carried out by amplification refractory mutation system-polymerase chain reaction method followed by agarose gel electrophoresis. RESULTS: The frequency of 5A/5A, 5A/6A, and 6A/6A genotypes in GC patients were 7.69%, 76.92%, and 15.38%, while in controls were 5.31%, 86.73%, and7.96%, respectively. There was a significant difference in the distribution of 5A/6A genotype in patients compared to the controls (P < 0.05). Conclusion: This study showed an increased frequency of heterozygotes for stromelysin-1 rs3025058 and thought to be involved in the etiology of GC.


Keywords: Gastric cancer, matrix metalloprotienases, matrix metalloprotienases-3, polymorphism, risk factors rs3025058, stromelysin-1


How to cite this article:
Krishnaveni D, Bhayal A C, Shravan K P, Jyothy A, Pratibha N, Venkateshwari A. Heterozygosity of stromelysin-1 (rs3025058) promoter polymorphism is associated with gastric cancer. Indian J Cancer 2015;52:251-4

How to cite this URL:
Krishnaveni D, Bhayal A C, Shravan K P, Jyothy A, Pratibha N, Venkateshwari A. Heterozygosity of stromelysin-1 (rs3025058) promoter polymorphism is associated with gastric cancer. Indian J Cancer [serial online] 2015 [cited 2021 Aug 5];52:251-4. Available from: https://www.indianjcancer.com/text.asp?2015/52/2/251/175806



 » Introduction Top


Gastric cancer (GC), commonly referred to as stomach cancer, can develop in any part of the stomach and may spread throughout the stomach and to other organs; particularly the esophagus, lungs, lymph nodes, and the liver. On the whole 90% of all tumors of the stomach are cancerous and 95% of these malignancies account for gastric adenocarcinoma.[1] It is a complex and multifactorial disorder mediated by multiple genetic, epigenetic, and environmental risk factors.[2] It is the third most common cancer in India and the second leading site of cancer occurrence worldwide.[3] There is much variation in the incidence of GC globally. An increased incidence of GC was reported from Southern parts of Asia, mostly from China, Japan, and South Korea, and it was ascribed to the intake of long-term stored foods containing carcinogenic nitroso amine compounds.[4] GC has been accounted to be much frequent in males of South India and occurring a decade earlier than those of North India. The zonal differences in the occurrence of GC may be explained by the differences in dietary pattern and intake of tobacco and alcohol which act as potential risk factors.[2]

Matrix metalloproteinases (MMPs) compose a vast group of zinc dependent proteolytic endopeptidases encompassing a metal in their integral structure and thought to be involved in degeneration of proteins in the interstitial matrix including basement membrane and tissue remodeling through convoluted biological procedures.[5] MMPs perform a pivotal role in promoting proliferation of tumors, angiogenesis, cell migration, apoptosis, and connective tissue degradation.[6]

Stromelysin-1, popularly known as MMP-3 is one among the 20 plus MMPs, is supposed to have an impact on increasing the risk for novel tumor formation and up-regulated in different kinds of tumors.[7] The gene for stromelysin-1 is present in the MMP gene cluster localized on to chromosome11q22.3.[8] A single nucleotide polymorphism rs3025058 (5A/6A) resulting due to the insertion/deletion of an adenine at position −1171 of the stromelysin-1 gene promoter affects the binding of transcription factor and promoter activity.[9] The 5A allele has been shown to result in higher MMP-3 expression because of disrupted binding of a nuclear factor kappa B dimer, which acts as a transcriptional repressor,[10] while the 6A allele sequence has a stronger recognition for the repressor binding site than 5A. This results in enhanced transcription in the presence of the 5A allele.[11] Individuals with the 5A allele are susceptible to diseases like abdominal aortic aneurysm and acute myocardial infarction, attributed to enhanced gene expression.[12] The frequency of the 5A/5A genotype was increased in primary sclerosing cholangitis patients with ulcerative colitis compared to those without ulcerative colitis, but no association was observed.[13] An increased risk for breast cancer has been reported in carriers of 5A allele,[14] but was not confirmed by a subsequent study.[15] On the other hand, a significantly higher 6A/6A genotypes were presented in individuals with congenital anomalies such as cleft lip/palate than in controls.[16] Heterozygotes with 5A/6A genotype are thought to have the optimal balance of matrix accumulation and degradation.[17] There were no studies performed yet on the role of stromelysin-1 gene rs3025058 (−1171, 5A/6A promoter polymorphism) in GC. To explore whether the stromelysin-1 rs3025058 (−1171, 5A/6A promoter polymorphism) is involved in the etiology of gastric carcinogenesis, we carried out a case-control study investigating the association between the polymorphism and the risk for GC in South Indian population.


 » Materials and Methods Top


Study population

The present case–control study considered 208 endoscopically and histopathologically confirmed GC patients and 226 endoscopically confirmed healthy control subjects. All the GC patients have had the localized intestinal type of GC. The information on demographic features such as age, sex, dietary habits, weight, consanguinity, family history of cancer, addictions such as smoking and alcoholism was elicited along with an informed written consent from all the subjects using a structured questionnaire. All the patients were tested for Helicobacter pylori infectivity status in antral biopsies by urease test following the method of Vaira et al.[18]

Bio specimens

A total volume of 5 ml of blood was collected from each subject in vacutainers with and without anticoagulant for separation of plasma and serum, respectively. Genomic DNA was isolated from whole blood samples of all patients and control subjects following the salting out method of Lahiri and Nurnberger.[19]

Ethics

The study was approved by Ethics Committee of the Institute in order to conduct the experiments on human subjects and the procedures followed were in accordance with the ethical standards of the committee.

Stromelysin-1 rs3025058 (−1171, 5A/6A) genotyping by amplification refractory mutation system polymerase chain reaction

Analysis of rs3025058 (−1171, 5A/6A promoter polymorphism) of the stromelysin-1 gene was carried out by amplification refractory mutation system-polymerase chain reaction (PCR) method. The allele-specific primers used were as follows: Forward primer, 5'-GAT TAC AGA CAT GGG TCA CGG CAC-3', and reverse primer, 5'-AAT CAG GAC AAG ACA TGG TTT TTC-3' for the 5A allele or 5'-AAT CAG GAC AAG ACA TGG TTT TTT-3' for the 6A allele. The amplification protocol consisted of an initial denaturation step at 95°C for 5 min. After this, each cycle consisted of three steps (94°C for 30 s, 61°C for 30 s and 72°C for 1 min). The cycle was repeated 30 times, followed by an additional extension at 72°C for 5 min.

Agarose gel electrophoresis

After performing PCR, the amplicons were resolved on a 3% agarose gel stained with ethidium bromide and visualized under ultraviolet gel documentation. [Figure 1] The presence of homozygous alleles at −1171 was noticed by the presence of 129 bp DNA bands while the heterozygotes displayed the presence of two bands. The genotypes were determined based on the appearance of bands with the help of 100 bp ladder. Ten percent of the samples were taken randomly, subjected to sequencing and no bias observed in the genotyping. The study revealed similar findings with 100% concordant results.
Figure 1: Gel picture showing the various genotypes of stromelysin-1 polymorphism

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Statistical analyses

The evaluation of the case and control differences in the distribution of alleles and genotypes was carried out by χ2 test of association. Odds ratios (ORs) and corresponding 95% confidence intervals were determined using Javastat 2-way contingency table analysis to measure the strength of association between stromelysin-1 gene polymorphism and GC.[20] All the statistical tests were two-tailed, and P values were considered to be statistically significant at P < 0.05 level of significance.[20]


 » Results Top


The present case–control study included a total of 208 GC patients and 226 control subjects. The demographic features of the population under study had been listed in [Table 1]. The patients were categorized based on sex, age, family history, H. pylori infection, addictions such as smoking and alcoholism, etc., The risk factor profile of the patients revealed that male sex, age above 50 years, smoking and alcoholism were the most common risk factors in patients compared to the control subjects (P < 0.05). Among the patients, 7.7% of them revealed familial incidence of GC. There was a significant difference between cases and controls with regard to consanguinity (P = 0.00) and H. pylori infection (P = 0.00).
Table 1: Demographic details of GC patients and control subjects

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The frequency of 5A/5A, 5A/6A, and 6A/6A genotypes of stromelysin-1 gene rs3025058 in GC patients were 7.69%, 76.92%, and 15.38%, while in controls were 5.31%, 86.73%, and 7.96%, respectively. The 5A and 6A allelic frequencies were 0.46 and 0.54 in the patient group, whereas 0.49 and 0.51 in controls, respectively. The genotype distribution was not in Hardy Weinberg equilibrium in both cases and controls.

[Table 2] displays the odds risk estimates of the genotypes in GC patients compared to control subjects There was a significant difference in the distribution of 5A/6A genotype (5A/6A vs. 6A/6A, χ2 = 5.645; P = 0.015; OR = 0.459 [0.237–0.883]) and (5A/6A vs. 5A/5A + 6A/6A, χ2 = 6.414; P = 0.009; OR = 0.510 [0.300–0.867]) in the patient group compared with the control subjects exhibiting 50% increased risk with 5A/6A genotype in patients. There was a significant association of 5A/6A genotype (P < 0.05) with all the categories such as age, sex, diet, alcohol, smoking, consanguinity, and familial incidence indicating an association of heterozygosity with an increased risk for GC. However surprisingly, 6A/6A genotype, revealed the association with an advanced age (P = 0.038) and alcoholism (P = 0.016).
Table 2: Odds risk estimates of the genotypes in GC patients compared with control subjects

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 » Discussion Top


Gastric cancer is a multifactorial disorder resulting from distinctive genetic and epigenetic predisposition and effect of environmental risk factors.[2] MMPs are important in altering the cell cycle checkpoint controls, thus promoting genomic imbalance by influencing cell adhesion and commit to initiation and development of tumors by transforming the cellular micro environment that help tumor formation.[6] As the stromelysin-1 rs3025058 (−1171, 5A/6A) polymorphism is located in the promoter region of the gene, it effects the regulation of the gene expression at transcriptional level.

This study revealed the significant association of demographic factors such as advanced age, male sex, smoking, alcoholism in the patient group compared to the control subjects. It is evident that most of the patients belonged to age above 50 years showcasing GC incidence to be higher in advanced age group compared with the early age group and is in agreement with the study of Neugut et al.[21] It is clearly observed that risk of developing GC is higher in males in comparison to females and the same is declared by Parkin et al.[22] This may be assigned to environmental risk triggering factors like alcohol and smoking, which is observed more common in males. The cytoprotective effect of estrogen hormone may be one of the factors for lower frequency of GC in women. Smoking and alcoholism were identified as potential risk factors in the development of GC. Earlier studies also shown that smoking and alcohol were cooperating in enhancing GC risk, which is correlated with the present findings and interpreted based on the fact that cigarette smoke may increase the risk to develop GC through the formation of nitroso amine, a potent carcinogen, while consuming alcohol had an impact on tumor volume doubling time invigorating tumor growth by promoting angiogenesis.[23]

Stromelysin-1 rs3025058 polymorphism was analyzed in several case–control studies from different ethnic groups and revealed contradicting results. There were no studies yet, from Indian context on the role of stromelysin-1 gene (−1171) promoter rs3025058 5A/6A polymorphism in GC. In this study, the genotype distribution disclosed a significant association of 5A/6A genotype in GC patients compared to the controls exhibiting increased risk for GC development, while the 6A/6A genotype acting as a risk factor in combination with an advanced age and alcoholism in patients causing two-fold increase in development of the disease. The contradictory results obtained in the current study may be explained by a most striking observation that there is a difference in the frequencies of 5A and 6A alleles across various ethnic groups, with 5A alleles being much more common in Caucasian groups. Conversely, the frequency of the 5A allele of the stromelysin-1 gene promoter appears to be low in Asian population as per the meta-analysis conducted by Yang et al.[24]


 » Conclusion Top


The present data provides the first molecular epidemiological evidence from Indian cohort for the association of stromelysin-1 rs3025058 (−1171, 5A/6A) promoter polymorphism with a risk to develop GC and thus suggesting the indirect role of stromelysin-1 polymorphism in altering cellular micro environment and facilitation of tumor development. Based on the conflicting results of these association studies, it is possible to assume that the polymorphism may influence the etiology of the GC, but this impact may be slight and perhaps restricted to some specific environment-genotype effect. A large confirmatory study involving other populations is warranted to understand the population-specificity and the relative contribution of this polymorphism in the disease phenotype.

 
 » References Top

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Schwartz GK. Invasion and metastases in gastric cancer:In vitro and in vivo models with clinical correlations. Semin Oncol 1996;23:316-24.  Back to cited text no. 1
    
2.
Krishnaveni D, Bhayal AC, Sri Manjari K, Vidyasagar A, Uma Devi M, Ramanna M, et al. MMP 9 gene promoter polymorphism in gastric cancer. Indian J Clin Biochem 2012;27:259-64.  Back to cited text no. 2
    
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Sumathi B, Ramalingam S, Navaneethan U, Jayanthi V. Risk factors for gastric cancer in South India. Singapore Med J 2009;50:147-51.  Back to cited text no. 3
    
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Sternlicht MD, Lochter A, Sympson CJ, Huey B, Rougier JP, Gray JW, et al. The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis. Cell 1999;98:137-46.  Back to cited text no. 7
    
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Spurr NK, Gough AC, Gosden J, Rout D, Porteous DJ, Heyningen VV, et al. Restriction fragment length polymorphism analysis and assignment of the metalloproteinases stromelysin and collagenase to the long arm of chromosome 11. Genomics 1988:2:119-27.  Back to cited text no. 8
    
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Ye S, Watts GF, Mandalia S, Humphries SE, Henney AM. Preliminary report: Genetic variation in the human stromelysin promoter is associated with progression of coronary atherosclerosis. Br Heart J 1995;73:209-15.  Back to cited text no. 9
    
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Borghaei RC, Rawlings PL Jr, Javadi M, Woloshin J. NF-kappaB binds to a polymorphic repressor element in the MMP-3 promoter. Biochem Biophys Res Commun 2004;316:182-8.  Back to cited text no. 10
    
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Ye S. Polymorphism in matrix metalloproteinase gene promoters: Implication in regulation of gene expression and susceptibility of various diseases. Matrix Biol 2000;19:623-9.  Back to cited text no. 11
    
12.
Terashima M, Akita H, Kanazawa K, Inoue N, Yamada S, Ito K, et al. Stromelysin promoter 5A/6A polymorphism is associated with acute myocardial infarction. Circulation 1999;99:2717-9.  Back to cited text no. 12
    
13.
Wiencke K, Louka AS, Spurkland A, Vatn M, Schrumpf E, Boberg KM, et al. Association of matrix metalloproteinase-1 and-3 promoter polymorphisms with clinical subsets of Norwegian primary sclerosing cholangitis patients. J Hepatol 2004;41:209-14.  Back to cited text no. 13
    
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Ghilardi G, Biondi ML, Caputo M, Leviti S, DeMonti M, Guagnellini E, et al. A single nucleotide polymorphism in the matrix metalloproteinase-3 promoter enhances breast cancer susceptibility. Clin Cancer Res 2002;8:3820-3.  Back to cited text no. 14
    
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Lei H, Zaloudik J, Vorechovsky I. Lack of association of the-1171 (5A) allele of the MMP3 promoter with breast cancer. Clin Chem 2002;48:798-9.  Back to cited text no. 15
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Letra A, Silva RA, Menezes R, Astolfi CM, Shinohara A, de Souza AP, et al. MMP gene polymorphisms as contributors for cleft lip/palate: Association with MMP3 but not MMP1. Arch Oral Biol 2007;52:954-60.  Back to cited text no. 16
    
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Medley TL, Kingwell BA, Gatzka CD, Pillay P, Cole TJ. Matrix metalloproteinase-3 genotype contributes to age-related aortic stiffening through modulation of gene and protein expression. Circ Res 2003;92:1254-61.  Back to cited text no. 17
    
18.
Vaira D, Holton J, Cairns S, Polydorou A, Falzon M, Dowsett J, et al. Urease tests for Campylobacter pylori: Care in interpretation. J Clin Pathol 1988;41:812-3.  Back to cited text no. 18
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Lahiri DK, Nurnberger JI Jr. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res 1991;19:5444.  Back to cited text no. 19
    
20.
Java Stat 2-way Contingency Table Analysis. Available from: http://www.statpages.org/ctab2×2.html. [Last accessed on 2014 Feb 08].  Back to cited text no. 20
    
21.
Neugut AI, Hayek M, Howe G. Epidemiology of gastric cancer. Semin Oncol 1996;23:281-91.  Back to cited text no. 21
    
22.
Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74-108.  Back to cited text no. 22
    
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Sung NY, Choi KS, Park EC, Park K, Lee SY, Lee AK, et al. Smoking, alcohol and gastric cancer risk in Korean men: The National Health Insurance Corporation Study. Br J Cancer 2007;97:700-4.  Back to cited text no. 23
    
24.
Yang X, Hu JW, Qiu MT, Li M, Yin R, Wang J, et al. Association of matrix metalloproteinase-3-1171 (5A>6A) polymorphism with cancer risk: A meta-analysis of 41 studies. PLoS One 2014;9:e87562.  Back to cited text no. 24
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2]

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