Relação entre Helicobacter pylori, metilação gênica e polimorfismos genéticos do hospedeiro no câncer gástrico

Abstract

Gastric cancer is a serious health problem. Several risk factors have been identified to contribute to its development, such as virulence genes of H. pylori, SNPs in host’s key genes and epigenetic events. The aim of this study was to investigate the relationship between genes methylation in gastric cancer, its association with H. pylori virulence genes, and verify the influence of CDH1 and MLH1 SNPs in the methylation status and correlate methylation of the promoter of miR-34b/c expression of MET protein in those cases. The promoter genes methylation was assessed by MS-PCR and HRM (for miR-34b/c) techniques. H.pylori genes were detected by PCR and SNPs, by PCR-RFLP. H.pylori genes were detected by PCR and SNPs, by PCR-RFLP. The genes of higher and lower methylation frequency were MSH2 and MLH1, respectively. Patients aged ≥ 50 had CDH1 more frequently methylated than those younger. The MLH1 gene was significantly less methylated in cardia tumors. As for the TNM, the CDH1-M gene was associated with advanced tumor extension, even when subdivided by subtype, since same association has been shown in tumors of intestinal subtype. Already the combination CDKN2A-M/MLH1-U was associated with absence of distant metastases. Taking into account the virulence genes of H. pylori, it was seen that patients infected vacAm1+ or cagG+ strains had a higher frequency of CDKN2A-U vacAs1 + was associated with COX-2-U and CDH1-U, while hopQII with MLH1-U. Patients infected with cagE+ and virB11+ strains had higher frequency of MSH2-M. It was also observed that less virulent strains tended to non-methylation for CDH1 and MLH1 genes. The classification trees made according to the histological subtype, showed that in tumors of diffuse subtype cagE+ and virB11 + strains (separately or together) were also attached to a methylator character of some of the analyzed genes. Already strains virB11+cagA+ did not lead to methylation in two genes. In intestinal subtype tumors was not observed a pattern having a variation in the combinations of H. pylori genes leading to the methylation. CDH1 SNPs presented no relation to the methylation of this gene, while the GA genotype of MLH1 SNP showed a tendency to not methylation of the gene. As regards the promoter methylation of miR-34b/c, the distribution percentage of the cases showed that the majority of cases presented methylation above 50%. The larger frequency among tumors of the intestinal subtype was in the methylation range of 50-75%, and to diffuse subtype from 75%. Already c-MET expression was positive in 86.36% of cases were not found differences among the different locations and subtypes. There was no correlation between the methylation percentage of miR-34b/c and c-MET expression. Observing each case, it was seen that in most cases whose c-MET expression was negative (H-score 0) had a high percentage of methylation of miR-34b/c. In conclusion, the methylation status of the studied genes appear to be dependent on the tumor location and is also related to the TNM staging and tumor subtype. In addition, more virulent H. pylori strains may be involved in this process by increasing inflammatory response leading to methylation of these genes. Already c-MET is overexpressed in cases of gastric cancer and the promoter region of miR-34b/c hipermethylated, indicating a possible involvement of these factors in carcinogenesis of the stomach, although it was not found a direct relationship between the two variables.