Association between Genetic Instability and Helicobacter pylori Infection in Gastric Epithelial Dysplasia

Abstract

Background. In gastric carcinogenesis, changes of DNA methylation appear to be an early molecular event, and the genome-wide methylation state is closely correlated with the level of long interspersed nucleotide element-1 (LINE-1) methylation. In this study, we measured LINE-1 methylation level according to genetic instability and evaluated the effect of Helicobacter pylori infection on genetic instability in gastric epithelial dysplasia. Methods. Total 100 tissue samples of gastric epithelial dysplasia were analyzed. Seven loci that linked to tumor suppressor genes were used to identify significant structural chromosomal aberrations. Microsatellite status was investigated for two different microsatellite marker loci (BAT25 and BAT26). Also, we measured LINE-1 methylation level by combined bisulfite restriction analysis (COBRA-LINE-1) method. Results. There were no significant differences of LINE-1 methylation level according to chromosomal/microsatellite instability and H. pylori state. In the dysplastic lesions with H. pylori infection, LINE-1 methylation level of MSI lesion was significantly lower than that of microsatellite stable (MSS) lesion (40.23 ± 4.47 versus 43.90 ± 4.81%, P < 0.01). Conclusions. In gastric epithelial dysplasia with H. pylori infection, MSI is correlated with reduced LINE-1 methylation level. Coexistence of H. pylori infection and MSI might be a driving force of gastric carcinogenesis.

Figure 1

Representative example of loss of heterozygosity (LOH) and microsatellite instability (MSI). Seven loci that linked to tumor suppressor genes were used to identify significant structural chromosomal aberrations (D5S505, D10S501, D10S602, TP53, D17S855, D18S58 and D18S61). Microsatellite status was investigated for two different microsatellite marker loci (BAT25 and BAT26). (NL: normal; T: tumor).

Figure 2

Assessment of LINE-1 hypomethylation status by COBRA LINE-1 method. Calculation was based on the ratio of the digested bands divided by the sum of the digested and undigested bands as described in Materials and Methods section (N: normal; T: tumor).

Figure 3

Level of LINE-1 hypomethylation of gastric epithelial dysplasia according to H. pylori state and genetic instability. (a) Irrespective of H. pylori infection, there were no differences of LINE-1 methylation level between LOH-L and LOH-H. (b) In dysplasia with H. pylori infection, LINE-1 methylation level of MSI is significantly reduced than that of MSS. Box plots illustrate median values, 25th and 75th percentiles, and outliers on a linear scale. The unpaired t test was applied for nonparametric statistical analysis, and *was considered statistically significant (P < 0.05).

Figure 4

Level of LINE-1 hypomethylation in gastric epithelial neoplasias categorized by the revised Vienna classification. There were no significant differences of LINE-1 methylation level according to the degree of LOH and MSI state. Except in category 3, the lesions with MSI had the lower LINE-1 methylation level than that of MSS. Box plots illustrate median values, 25th and 75th percentiles, and outliers on a linear scale. The unpaired t test and one way ANOVA were applied for nonparametric statistical analysis, and *was considered statistically significant (P < 0.05).