Comparative genomic study of gastric epithelial cells co-cultured with Helicobacter pylori

Abstract

Aim: To identify genes potentially involved in Helicobacter pylori (H. pylori)-induced gastric carcinogenesis.

Methods: GES-1 cells were co-cultured with H. pylori strains isolated from patients with gastric carcinoma (GC, n = 10) or chronic gastritis (CG, n = 10) for in vitro proliferation and apoptosis assays to identify the most and least virulent strains. These two strains were cagA-genotyped and used for further in vivo carcinogenic virulence assays by infecting Mongolian gerbils for 52 wk, respectively; a broth free of H. pylori was lavaged as control. Genomic profiles of GES-1 cells co-cultured with the most and least virulent strains were determined by microarray analysis. The most differentially expressed genes were further verified using quantitative real-time polymerase chain reaction in GES-1 cells infected with the most and least virulent strains, and by immunohistochemistry in H. pylori positive CG, precancerous diseases, and GC biopsy specimens in an independent experiment.

Results: GC-derived H. pylori strains induced a potent proliferative effect in GES-1 cells in co-culture, whereas CG-derived strains did not. The most (from a GC patient) and least (from a CG patient) virulent strains were cagA-positive and negative, respectively. At week 52, CG, atrophy, metaplasia, dysplasia, and GC were observed in 90.0%, 80.0%, 80.0%, 90%, and 60.0%, respectively, of the animals lavaged with the most virulent strain. However, only mild CG was observed in 90% of the animals lavaged with the least virulent strain. On microarray analysis, 800 differentially expressed genes (49 up- and 751 down-regulated), involving those associated with cell cycle regulation, cell apoptosis, cytoskeleton, immune response, and substance and energy metabolisms, were identified in cells co-cultured with the most virulent strain as compared with those co-cultured with the least virulent strain. The six most differentially expressed genes (with a betweenness centrality of 0.1-0.2) were identified among the significant differential gene profile network, including JUN, KRAS, BRCA1, SMAD2, TRAF1, and HDAC6. Quantitative real-time polymerase chain reaction analyses verified that HDAC6 and TRFA1 mRNA expressions were significantly more up-regulated in GES-1 cells co-cultured with the most virulent strain than in those co-cultured with the least virulent strain. Immunohistochemistry of gastric mucosal specimens from H. pylori-positive patients with CG, intestinal metaplasia (IM), dysplasia, and GC showed that moderately positive and strongly positive HDAC6 expression was detected in 21.7% of CG patients, 30.0% of IM patients, 54.5% of dysplasia patients, and 77.8% of GC patients (P < 0.001). The up-regulation of TRAF1 expressions was detected in 34.8%, 53.3%, 72.7%, and 88.9% specimens of CG, IM, dysplasia, and GC, respectively (P < 0.001).

Conclusion: The overexpression of HDAC6 and TRAF1 in GES-1 cells co-cultured with the GC-derived strain and in H. pylori-positive dysplasia and GC suggests that HDAC6 and TRAF1 may be involved in H. pylori-induced gastric carcinogenesis.

Keywords: Gastric carcinoma; Genomic profiles; Helicobacter pylori; Proliferation.

Figure 1

GES-1 cell proliferation as determined by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide assay (A) and cell apoptosis as determined by flow cytometry (B) after co-culture with helicobacter pylori isolated from patients with gastric carcinoma (GC, n = 10) or chronic gastritis (CG, n = 10) for 48 h in relation to control cells. The ratio denotes the ratio of GES-1 cells vs Helicobacter pylori cells. ^aP < 0.05 vs control group; ^cP < 0.05 vs CG group. OD: Optical density.

Figure 2

Identification of status of cagA gene in Helicobacter pylori strains using real-time polymerase chain reaction. A: DNA ladder; B: Helicobacter pylori (H. pylori) stain NCTC 11639 (positive control); C: The most virulent gastric carcinoma-derived strain; D: The least virulent chronic gastritis-derived strain; E: H. pylori stain NCTC 12908 (negative control).

Figure 3

Representative histological microphotographs (hematoxylin and eosin staining, 200 ×, scale bar = 100 μm) of gastric mucosal specimens collected from Mongolian gerbils lavaged with the GC-derived Helicobacter pylori strain (A: gastric carcinoma), the CG-derived strain (B: chronic gastritis), and the control lixivium (C) at the 28th week.

Figure 4

Cluster analysis of gene expression profiles in GES-1 cells in response to the most and least virulent Helicobacter pylori strains. The red, green, and black colors indicate up-regulated, down-regulated, and equivalent expression. 1-1: Uninfected GES-1 cells (control); 2-2: GES-1 cells co-cultured with the GC-derived strain; 3-2: GES-1 cells co-cultured with the CG-derived strain. GC: Gastric carcinoma; CG: Chronic gastritis.

Figure 5

Verification of HDAC6 and TRAF1 up-regulation in GES-1 cells in response to the most and least virulent Helicobacter pylori strains as determined by quantitative real-time polymerase chain reaction analysis. A: Representative quantitation of mRNA expression of GAPDH, HDAC6 and TRAF1 in GES-1 cells (a), GES-1 cells co-cultured with the CG-derived Helicobacter pylori (H. pylori) strain (b) and GES-1 cells co-cultured with the GC-derived H. pylori strain (c); B: Graphic analysis showing mRNA expression of HDAC6 and TRAF1 expressions in GES-1 cells in GES-1 cells, GES-1 cells co-cultured with the CG-derived H. pylori strain and GES-1 cells co-cultured with the GC-derived H. pylori strain. ^bP < 0.01 vs the control; ^dP < 0.01 vs cells co-cultured with the CG-derived H. pylori strain.

Figure 6

Histology and immunohistochemistry (400 ×, scale bar = 50 μm) of HDAC6 and TRAF1 in gastric specimens of patients with chronic gastritis (CG, n = 23), intestinal metaplasia (IM, n = 30), dysplasia (n = 33), and gastric carcinoma (GC, n = 27).