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Association between Virulence Factors and TRAF1/4-1BB/Bcl-xL Expression in Gastric Mucosa Infected with Helicobacter pylori

Objective. CagA+/vacAs1+/vacAm1+ Helicobacter pylori upregulates the expression of tumor necrosis factor receptor–associated factor 1 (TRAF1), tumor necrosis factor receptor superfamily member 9 (4-1BB), and B-cell lymphoma-extra large (Bcl-xL) in human gastric epithelial cells. We investigated the correlation between cagA/vacAs1/vacAm1 and TRAF1/4-1BB/Bcl-xL expression in gastric mucosal tissue of patients with gastric disorders. Methods. We collected gastric mucosa samples from 35 chronic, nonatrophic gastritis (CG) patients, 41 atrophic gastritis patients, 44 intestinal metaplasia with atypical hyperplasia (IM) patients, and 28 gastric carcinoma (Ca) patients. The expression of  TRAF1, 4-1BB, and Bcl-xL was determined using western blotting. The expression of cagA, vacAs1, and vacAm1 in H. pylori was examined with polymerase chain reaction. Results. The expression of TRAF1, 4-1BB, and Bcl-xL was significantly upregulated in IM and Ca patients (P < 0.05 compared with CG). There were more cases of cagA+/vacAs1+/vacAm1+ H. pylori infection in samples with elevated TRAF1, 4-1BB, or Bcl-xL expression (P < 0.05). Additionally, there were a remarkably large number of samples with upregulated TRAF1/4-1BB/Bcl-xL expression in cases of cagA+/vacAs1+/vacAm1+ H. pylori infection (44 cases, 67.7%; P < 0.05). Conclusions. The pathogenesis of IM and Ca may be promoted by cagA+/vacAs1+/vacAm1+ H. pylori, possibly via upregulated TRAF1, 4-1BB, and Bcl-xL in gastric mucosal tissue.


Introduction
Helicobacter pylori, a gram-negative bacterium present in nearly 50% of the global population [1,2], is one of the main causes of peptic ulcer disease [3]. In addition, H. pylori infection is related to gastric carcinoma, possibly inducing chronic gastritis, and progresses to the premalignant stages of atrophic gastritis, intestinal metaplasia, and, eventually, gastric carcinoma [4][5][6]. However, knowledge of the pathogenesis of H. pylori infection and gastric diseases is incomplete.
H. pylori strains that express virulence genes such as cagA and vacA are linked to increased risk of gastric cancer [7]. Previously, we performed a comparative genomic study of gastric epithelial cells cocultured with H. pylori and found that the expression of tumor necrosis factor receptor-associated factor 1 (TRAF1), 4-1BB (also known as CD137), and B-cell lymphoma-extra large (Bcl-xL) was significantly upregulated in human gastric epithelial GES-1 cells infected with H. pylori expressing the virulence genotype cagA+/vacAs1+/vacAm1+ [8]. TRAF1 expression in human gastric mucosa is related to the H. pylori virulence genotype cagA+/vacAs1+/vacAm1+ [9], whereas the correlation of 4-1BB and Bcl-xL gene expression with cagA, vacAs1, and vacAm1 toxicity at different stages of gastric disease have not been studied.

Western Blotting.
Total protein was extracted from the tissue, and protein concentrations were measured using a bicinchoninic acid protein assay kit according to the manufacturer's instructions (Beijing CoWin Biotech). Equal amounts of protein extracts were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The PVDF membranes were blocked before they were incubated with primary antibodies at 4 ∘ C overnight. The primary antibodies used were anti-TRAF1 (1 : 500), anti-4-1BB (1 : 500), anti-Bcl-xL (1 : 1000), and anti-GAPDH (1 : 5000). After washing, the membranes were incubated with goat anti-rabbit or antimouse IgG HRP-labeled secondary antibodies (1 : 5000). Immunobands were visualized using an enhanced chemiluminescence kit according to the manufacturer's instructions (Beijing CoWin Biotech). The densitometric values of the immunobands were used for statistical analysis. GAPDH was used as the internal control. Data were quantified from at least three independent experiments.

Polymerase Chain Reaction.
Total DNA was extracted from H. pylori-infected gastric mucosal tissue using a General AllGen Kit for genomic DNA extraction according to the manufacturer's instructions (Beijing CoWin Biotech). The purity and concentration of the extracted DNA was measured using an ultraviolet spectrometer with an optical density (OD) 260/OD280 ratio. The primers for the H. pylori virulence factors were designed as described previously [10]. The forward and reverse primers and PCR product length are listed in Table 1. The PCR conditions were as follows: initial denaturation at 95 ∘ C for 5 min; denaturation at 95 ∘ C for 30 s; annealing at different temperatures (57 ∘ C (cagA), 52 ∘ C (vacAs1), 56 ∘ C (vacAm1)) for 30 s; and extension at 72 ∘ C for 40 s for a total of 39-40 cycles, followed by a final extension at 72 ∘ C for 7 min [11].

Statistical
Analysis. Data were analyzed using SPSS 17.0 software (IBM, New York, NY, USA). Measurement data are presented as the means ± standard deviation. Comparison among measurement data in different groups was conducted with diverse mean comparison of analysis of variance. A chi-square test was used for comparing enumeration data from different groups. Data correlation was analyzed using Spearman's analysis. < 0.05 was considered significantly different.

TRAF1, 4-1BB, and Bcl-xL Expression in Gastric Mucosal
Tissue. First, we examined TRAF1, 4-1BB, and Bcl-xL protein expression in gastric mucosal tissue from subjects with CG, AG, IM, and Ca using western blotting. As seen in Figure 1, TRAF1, 4-1BB, and Bcl-xL levels were significantly
TRAF1 belongs to a group of structurally similar adapter proteins (TRAFs), but differs from other TRAFs because it lacks the conserved N-terminal RING domain found in other TRAF family proteins [12]. TRAF1 plays a critical role in regulating apoptosis by indirectly modulating the transcription factor nuclear factor-B inducible gene expression [12]. In a previous study, we found that TRAF1 was upregulated in several gastric cancer cell lines, including BGC823, SGC7901, and MGC803 [13]. Moreover, TRAF1 expression in human gastric mucosa is related to the H. pylori virulence genotype cagA+/vacAs1+/vacAm1+ [9]. In accordance with these findings, we demonstrate here that TRAF1 was highly expressed in the gastric mucosa of IM and Ca patients and that TRAF1 upregulation correlated positively with the H. pylori cagA+/vacAs1+/vacAm1+ virulence genotype.
Bcl-xL and 4-1BB are two key regulators in the TRAF1 signaling pathway. TRAF1 has a prosurvival effect in CD8 T cells via the 4-1BB-mediated upregulation of Bcl-xL [14]. Bcl-xL, an antiapoptotic member of the Bcl-2 family, is involved in modulating the angiogenic phenotype of human tumor cells [15,16]. Via cross-talk with P-glycoprotein, Bcl-xL acts as an antiapoptotic factor in H. pylori-related gastric carcinogenesis [17]. Hence, we investigated 4-1BB and Bcl-xL expression in the gastric mucosa from different gastric diseases. 4-1BB expression was greatly upregulated in AG, IM, and Ca gastric mucosa, and Bcl-xL levels were increased, especially in IM and Ca gastric mucosa. Consistent with our findings, Yang et al. reported that Bcl-xL expression was relatively low in CG and AG patients, but was markedly increased in Ca patients [18]. As TRAF1, 4-1BB, and Bcl-xL are correlated positively, TRAF1 may trigger 4-1BB-mediated Bcl-xL activity. TRAF1-4-1BB-Bcl-xL signaling pathway upregulation may play a critical role in the development gastritis into gastric cancer.
A significantly high prevalence of East Asian cagApositive H. pylori infection has been reported in gastric cancer patients (84.6%), suggesting that cagA-positive H. pylori infection and gastric cancer are closely associated [19]. Furthermore, it has been suggested that the vacAs1+/vacAm1+ genotype is associated with gastric cancer [20]. Importantly, a high proportion of subjects with upregulated TRAF1, 4-1BB, and Bcl-xL expression in the present study were infected with cagA+/vacAs1+/vacAm1+ H. pylori. In contrast, Matsumoto   et al. showed that H. pylori vacA reduced Bcl-xL expression in gastric adenocarcinoma cell lines and led to apoptosis [21]. This discrepancy might be due to the differences between in vitro and in vivo conditions. Additionally, we cannot discount the possibility that differing virulence genotypes (cagA+/vacAs1+/vacAm1+, vacA+) may have different effects. Future studies should continue to investigate the effects of the cagA+/vacAs1+/vacAm1+ virulence genotype on TRAF1, 4-1BB, and Bcl-xL expression in cultured gastric cancer cells.
In summary, our study implies that cagA+/vacAs1+/ vacAm1+ H. pylori infection might promote gastritis progression to gastric cancer, possibly by upregulating TRAF1, 4-1BB, and Bcl-xL expression in gastric mucosal tissue. It is possible that cagA+/vacAs1+/vacAm1+ H. pylori upregulates TRAF1 activation, which triggers 4-1BB-mediated Bcl-xL activation, thereby exerting an antiapoptotic effect and contributing to the pathogenesis of gastric cancer. Nevertheless, the underlying mechanism involved in this process requires further clarification.