Interaction Of Helicobacter Pylori With MDM2 SNP309 Polymorphisms And Susceptibility To Gastric Cancer Studies

Background & Aims:Gastric cancer is the common cancer in the world and particularly prevalent in certain countries including China. H. pylori is a definite careinogen for gastric adenocarcinoma. However, the underlying pathogenic mechanisms have not been fully understood, although the interactions between environmental, bacterial and multiple genetic components are likely to be involved. MDM2 (mouse double minute) is an oncogene, and can inhibit p53 passway. We hypothesized that genetic variations of MDM2(SNP309) was associated with gastric cancer susceptibility. To test this hypothesis, we performed genotyping analysis for MDM2 309T>G in a case–control study of 574 gastric cancer cases and 574 age and sex frequency-matched healthy controls from Han population of Jiangsu province.Methods: This study consisted of 574 patients with gastric cancer and 574 healthy controls. All subjects were unrelated ethnic Han Chinese and residents in Jiangsu Province. All cases were pathologically diagnosed as gastric carcinoma. The population controls were selected from cancer-free individuals living in Jiangsu Province, and were frequency matched to the cases on age (±5 years) and sex. Trained interviewers used a pre-tested questionnaire to determine demographic and lifestyle characteristics, such as sex, age, and related risk factors, including tobacco smoking, alcohol drinking, etc. MDM2 SNP309 was genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). The indirect solid phase immunochromatographic assay was used to investigate the presence of IgG antibodies to H. pylori (Genelabs Diagnostics Pty Ltd, Singapore). We used Western blotting analysis analyze association of the MDM2 SNP309 (T>G) polymorphism with expression levels of the MDM2 protein and immunohistochemistry was done in some of the genotyped gastric tissues to investigate the effect of the MDM2 SNP309 polymorphism on MDM2 expression. The luciferase assays in vitro various cell lines further compared SNP309G allele with the SNP309T allele. Kaplan-Meier survival curves was used to evaluate different genotype survival.Results:The cancer cases and controls seemed to be adequately matched in terms of sex and age. There was no difference between patients and controls in Hp seropositivity. The observed genotype frequencies of MDM2 polymorphism was in Hardy-Weinberg equilibrium in the controls (P>0.05). Distributions of MDM2 genotypes were then compared among patients and controls. The frequencies of MDM2 TT, TG, and GG genotypes among patients were significantly different from those among controls (P<0.05), with the GG homozygotes being overrepresented among patients compared with controls (24.6% versus 13.7%; P = 0.0005). The MDM2 GG genotype was associated with an increased risk of developing GC (OR=1.85; 95% CI, 1.31-2.61; P=0.0005), compared with TT genotype. However, the heterozygous genotype MDM2 TG was not associated with the risk. Significant interactions were observed between polymorphisms and Hp, with risk being the highest (OR=2.27; 95% CI, 1.58-3.26) in Hp seropositivity having MDM2 GG genotypes.Western blot shown that the MDM2 protein levels of individuals with the GG genotype were a significantly higher than that of those with the TT genotype (0.89±0.23 versus 0.28±0.07; P=0.02 for GG versus TT genotype, in arbitrary unites). The result of the immunohistochemistry: positive staining for MDM2 was detected in 2 of 13 (15.38%) TT genotype, 9 of 21 (42.86%) TG genotype, and 8 of 10 (80.00%) GG genotype carriers. The frequency of MDM2 expression in GG genotype carriers was significantly higher than that in TT genotype carriers (P=0.003). To evaluate the native promoter activity associated with the MDM2 SNP309 (T>G) polymorphism, we constructed luciferase reporter vectors (pGL3), spanning the 551 to 787 base from the first base of the gene, with either SNP309T or SNP309G allele and used them for transient transfections with the NIH-3T3, SGC-7901 and MKN28 cells. The vectors with the SNP309G allele had a 100% to 300% increase in the relative luciferase activities, compared with that of those with the SNP309G allele in all four types of cell lines (P <0.01 for all). Furthermore, we used LPS50ug (Helicobacter pylori extract) to stimulate MKN28 cells, whether wild or mutant, and the relative luciferase activity were significantly increased (P <0.05). These results suggested that the SNP309G allele in the promoter region indeed had an increased transcriptional activity of the gene MDM2. Additional Kaplan-Meier survival curves shown that SNP309 (G/G) was an independent marker of poor overall survival in gastric cancer.Conclusions:The MDM2 promoter SNP309 is associated with the presence of gastric carcinoma in Chinese patients especially with H. pylori infection.