Helicobacter pylori infection induced alteration of gene expression in human gastric cells

Abstract

Background: Helicobacter pylori, a human pathogen responsible for many digestive disorders, induces complex changes in patterns of gene expression in infected tissues. cDNA expression arrays provide a useful tool for studying these complex phenomena.

Aim: To identify genes that showed altered expression after H pylori infection of human gastric cells compared with uninfected controls.

Methods: The gastric adenocarcinoma cell line AGS was cocultivated with H pylori. Growth of infected cells was determined by trypan blue exclusion assay. Complementary DNA probes derived from H pylori treated and untreated cells were hybridised to two identical Atlas human cDNA expression arrays, and those genes with altered expression levels were identified. A real time quantitative reverse transcription-polymerase chain reaction assay was used to better define expression patterns of these genes in endoscopically gastric mucosal biopsies with and without H pylori infection.

Results: Over 24 hours, coincubation with H pylori inhibited AGS cell growth but did not cause a noticeable degree of cell death. H pylori treatment altered the pattern of gene expression in AGS cells. We identified 21 overexpressed genes and 17 suppressed genes from the cDNA expression arrays. The majority of genes were transcription factors such as c-jun, BTEB2, and ETR101. Other genes were involved in signal transduction pathways, such as MAP kinase, interleukin 5, and insulin-like growth factor. Genes involved in cell cycle regulation and differentiation, such as CDC25B and NM23-H2, were also identified. In patients with H pylori infection (n=20), there was a significant difference for ERCC3, Id-2, and NM23-H2 mRNA levels in infected gastric mucosa compared with uninfected gastric mucosa in patients without peptic diseases (n=20) (ERCC3 4.75 molecules/10(4) beta-actin mRNA molecules v 13.65, p<0.001; Id-2 16.1 v 23.4, p<0.05; NM23-H2 17.5 v 45.5, p<0.001). There was no significant difference between mRNA levels of c-jun and CDC25B in H pylori colonised gastric mucosa and uninfected mucosa.

Conclusion: We demonstrated that H pylori infection caused alteration of gene expression in AGS cells. The differential hybridisation technique of Atlas human cDNA expression array is a useful method to identify host genes involved in pathogenic mechanisms in H pylori infection.

Figure 1

Apoptosis fraction of AGS cells at different times after coculturing with H pylori. Synchronised AGS cells were serum deprived for 48 hours (time 0 hours) and fed with fresh medium containing 10% fetal bovine serum (FBS) alone or combined with wild-type H pylori at a bacterial concentration of 10⁷ cells/ml. The experiments were repeated three times.

Figure 2

Gene expression pattern after 24 hours of H pylori cocultured AGS cells appearing on the Atlas human cDNA expression arrays. Upper and lower arrays were hybridised with the cDNA probes derived from untreated and H pylori treated AGS cells, respectively. Some of the differentially expressed genes are marked. Open arrows indicate genes that were overexpressed by H pylori treatment; black arrows indicate downregulated genes. 1, c-jun; 2, BTEB2; 3, MAPk/ERK kinase; 4, α-catenin; 5, fibronectin receptor α subunit; 6, epidermal growth factor; 7, Id-2; 8, p55CDC; 9, nuclease sensitive element DNA binding protein; 10, NM23-H2; and 11, death associated protein 3. Internal controls were: 12, ubiquitin; 13, gapdh; 14, tubulin; 15, β-actin; 16, 23 kDa highly basic protein; and 17, ribosomal protein S9.

Figure 3

Northern blot demonstrating changes in expression of ERCC3, Id-2, NM23-H2, c-jun, and p55CDC at specified time intervals after coculturing AGS cells with H pylori at 10⁷ cells/ml for 24 hours. RNAs were purified at the indicated times, and 10 µg of total cellular RNA was loaded into each lane. The same blot was hybridised to one probe, retrieved, and rehybridised sequentially with the other probes. Three independent experiments were performed and similar result were obtained.

Figure 4

mRNA levels of ERCC3, Id-2, NM23-H2, c-jun, and p55CDC in human gastric mucosal biopsies. mRNA amounts were quantified using a real time quantitative reverse transcription-polymerase chain reaction technique, as described in patients and methods. For each group data were summarised using the box plot technique. The bottom and top symbols indicated minimum and maximal values, respectively (lower or higher than 10% and 90% percentiles); the filled square in the box indicates the mean; and the bottom of the box, median line, and top of the box indicate 25%, 50%, and 75% percentiles, respectively. + and − indicate H pylori infected and uninfected biopsies, respectively.