# Association between Virulence Factors andTRAF1/4-1BB/Bcl-xL Expression in Gastric Mucosa Infected with Helicobacter pylori

Authors: Fen Wang; Xiang Wu; Zhiying Liu; Guangkui Bu; Xiayu Li; Nanfang Qu; Jin Peng; Canxia Xu; Shourong Shen; Yi Yuan
Journal: Gastroenterology Research and Practice
 (2015)

---

## Abstract

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.


---

## Body

## 1. 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 [4–6]. However, knowledge of the pathogenesis ofH. pylori infection and gastric diseases is incomplete.H. pylori strains that express virulence genes such ascagA andvacA are linked to increased risk of gastric cancer [7]. Previously, we performed a comparative genomic study of gastric epithelial cells cocultured withH. pylori and found that the expression of tumor necrosis factor receptor–associated factor 1 (TRAF1),4-1BB (also known asCD137), and B-cell lymphoma-extra large (Bcl-xL) was significantly upregulated in human gastric epithelial GES-1 cells infected withH. pylori expressing the virulence genotypecagA+/vacAs1+/vacAm1+ [8]. TRAF1 expression in human gastric mucosa is related to theH. pylori virulence genotypecagA+/vacAs1+/vacAm1+[9], whereas the correlation of4-1BB andBcl-xLgene expression withcagA, vacAs1,andvacAm1 toxicity at different stages of gastric disease have not been studied.In the present study, we investigatedTRAF1, 4-1BB, andBcl-xL expression in gastric mucosa derived fromH. pylori–infected patients with chronic nonatrophic gastritis (CG), atrophic gastritis (AG), intestinal metaplasia with atypical hyperplasia (IM), and gastric carcinoma (Ca). We also determined the association betweenH. pylori virulence factors (cagA, vacAs1, vacAm1) andTRAF1/4-1BB/Bcl-xL expression at the different stages of gastric disease. Our findings may provide valuable insight into the pathogenesis and progression of chronic gastritis into gastric carcinoma.


## 2. Materials and Methods

### 2.1. Reagents

DNA marker and 2x Tag Master Mix were purchased from Beijing CoWin Biotech (Beijing, China). Polyvinyl difluoride (PVDF) membrane was obtained from Millipore (Billerica, MA, USA). PageRuler Prestained Protein Ladder was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies againstTRAF1andBcl-xL were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Millipore. Anti–4-1BB antibody was obtained from Abcam (Cambridge, MA, USA). Goat anti-rabbit and anti-mouse immunoglobulin G (IgG) horseradish peroxidase- (HRP-) conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Unless otherwise stated, all other reagents were provided by the Central South University Cancer Research Institute, Changsha, China.


### 2.2. Sample Collection

In total, 148 gastric antrum samples were collected from patients with different gastric mucosal diseases at the Gastroscope Room, The Third Xiangya Hospital of Central South University, Changsha, China, from January 2013 to December 2013.H. pylori infection was determined via rapid urease test, C13 urea breath test, and histological examination. All recruited subjects wereH. pylori positive;H. pylori–positive results were confirmed by at least two of the above tests. Among the patients, 35 had CG, 41 had AG, 44 had IM, and 28 had Ca. Subjects were recruited after they had signed an informed consent. Ethical approval for the study was granted by The Third Xiangya Hospital of Central South University.


### 2.3. 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 anti-mouse 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.


### 2.4. Polymerase Chain Reaction

Total DNA was extracted fromH. 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 theH. 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].Table 1
Primers and PCR conditions.
Gene
Primers (forward, reverse)
Annealing temperature (°C)
Amplified product length (bp)
cagA
5′-GATAACAGGCAAGCTTTTGAGG-3′
57
349
5′-CTGCAAAAGATTGTTTGGCAGA-3′
vacAs1
5′-ATGGAAATACAACAAACACAC-3′
52
259
5′-CTGCTTGAATGCGCCAAAC-3′
vacAm1
5′-CAATCTGTCCAATCAAGCGAG-3′
56
570
5′-GCGTCTAAATAATTCCAAGG-3′


### 2.5. 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.P
<
0.05 was considered significantly different.


## 2.1. Reagents

DNA marker and 2x Tag Master Mix were purchased from Beijing CoWin Biotech (Beijing, China). Polyvinyl difluoride (PVDF) membrane was obtained from Millipore (Billerica, MA, USA). PageRuler Prestained Protein Ladder was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies againstTRAF1andBcl-xL were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Millipore. Anti–4-1BB antibody was obtained from Abcam (Cambridge, MA, USA). Goat anti-rabbit and anti-mouse immunoglobulin G (IgG) horseradish peroxidase- (HRP-) conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Unless otherwise stated, all other reagents were provided by the Central South University Cancer Research Institute, Changsha, China.


## 2.2. Sample Collection

In total, 148 gastric antrum samples were collected from patients with different gastric mucosal diseases at the Gastroscope Room, The Third Xiangya Hospital of Central South University, Changsha, China, from January 2013 to December 2013.H. pylori infection was determined via rapid urease test, C13 urea breath test, and histological examination. All recruited subjects wereH. pylori positive;H. pylori–positive results were confirmed by at least two of the above tests. Among the patients, 35 had CG, 41 had AG, 44 had IM, and 28 had Ca. Subjects were recruited after they had signed an informed consent. Ethical approval for the study was granted by The Third Xiangya Hospital of Central South University.


## 2.3. 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 anti-mouse 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.


## 2.4. Polymerase Chain Reaction

Total DNA was extracted fromH. 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 theH. 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].Table 1
Primers and PCR conditions.
Gene
Primers (forward, reverse)
Annealing temperature (°C)
Amplified product length (bp)
cagA
5′-GATAACAGGCAAGCTTTTGAGG-3′
57
349
5′-CTGCAAAAGATTGTTTGGCAGA-3′
vacAs1
5′-ATGGAAATACAACAAACACAC-3′
52
259
5′-CTGCTTGAATGCGCCAAAC-3′
vacAm1
5′-CAATCTGTCCAATCAAGCGAG-3′
56
570
5′-GCGTCTAAATAATTCCAAGG-3′


## 2.5. 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.P
<
0.05 was considered significantly different.


## 3. Results

### 3.1.TRAF1,4-1BB, andBcl-xL Expression in Gastric Mucosal Tissue

First, we examinedTRAF1, 4-1BB,andBcl-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,andBcl-xL levels were significantly upregulated in IM and Ca gastric mucosa as compared with CG gastric mucosa (P
<
0.05). The expression of4-1BB was also elevated in AG gastric mucosa compared to CG gastric mucosa (P
<
0.05). Moreover, compared to AG gastric mucosa, there was an obvious increase inTRAF1 protein expression in Ca gastric mucosa (P
<
0.05). These findings show that the expression ofTRAF1, 4-1BB, and Bcl-xL was upregulated, especially in the gastric mucosa of IM and Ca patients in comparison to the other groups. Spearman’s analysis indicated that the expression ofTRAF1, 4-1BB,andBcl-xLwas positively correlated. TheTRAF1and4-1BB, TRAF1andBcl-xL,and4-1BBandBcl-xL correlation coefficient were 0.678, 0.702, and 0.694, respectively (P
<
0.05 for all three groups).Western blotting examination ofTRAF1, 4-1BB,andBcl-xL protein expression in gastric mucosal tissue from subjects with CG, AG, IM, or Ca. (a) Representative western blots. Relative expression of (b)TRAF1, (c)4-1BB, and (d)Bcl-xL was quantified against GAPDH. Data were quantified from three independent experiments. P
<
0.05
* compared with CG; #
P
<
0.05 compared with AG.
(a)
(b)
(c)
(d)


### 3.2. Analysis of Virulence Factors inH. pylori Extracted from Gastric Mucosa

We analyzedH. pylori virulence factors expression,that is, cagA, vacAs1,andvacAm1, inH. pylori extracted from the different gastric mucosal tissues. Representative positive expression of the virulence factors is presented in Figure 2. To clarify the association between theH. pylori virulence factors andTRAF1, 4-1BB,andBcl-xL expression, the number of samples positive or negative forcagA, vacAs1,andvacAm1was stratified according toTRAF1, 4-1BB, andBcl-xL expression (Table 2). According to the results of western blot, the gastric cancer and intestinal metaplasia with atypical hyperplasia groups were classified as theTRAF1, 4-1BB, andBcl-xL high expression group, while the gastritis and atrophic gastritis groups were classified as theTRAF1, 4-1BB, andBcl-xL low expression group. The number and percentage ofcagA-, vacAs1-,andvacAm1-positive cases were greatly upregulated in samples with highTRAF1, 4-1BB,orBcl-xL expression (P
<
0.05 compared with cases negative forcagA, vacAs1, vacAm1). In addition, the number ofcagA+/vacAs1+/vacAm1+ cases in samples with elevatedTRAF1, 4-1BB,orBcl-xLexpression was increased dramatically (P
<
0.05 compared with unalteredTRAF1, 4-1BB,orBcl-xLexpression).Table 2
Samples positive or negative forcagA, vacAs1, or vacAm1 stratified according toTRAF1, 4-1BB,and Bcl-xL expression.
Group
TRAF1
4-1BB
Bcl-xL
↑
—
↑
—
↑
—
cagA+/vacAs1+/vacAm1+
50 (64.9)*
15 (21.1)
50 (66.7)*
15 (20.5)
47 (59.4)*
18 (26.1)
cagA+/vacAs1+/vacAm1−
6 (7.8)
10 (14.1)
7 (9.3)
9 (12.3)
4 (5.1)
12 (17.4)
cagA+/vacAs1−/vacAm1+
3 (3.9)
12 (16.9)
3 (4.0)
12 (16.4)
7 (8.9)
8 (11.6)
cagA+/vacAs1−/vacAm1−
3 (3.9)
10 (14.1)
3 (4.0)
10 (13.7)
3 (3.8)
10 (14.5)
cagA−/vacAs1+/vacAm1+
3 (3.9)
6 (8.5)
5 (6.7)
4 (5.5)
5 (6.3)
4 (5.8)
cagA−/vacAs1+/vacAm1−
5 (6.5)
8 (11.3)
1 (1.3)
12 (16.4)
3 (3.8)
10 (14.5)
cagA−/vacAs1−/vacAm1+
2 (2.6)
5 (7.0)
3 (4.0)
4 (5.5)
4 (5.1)
3 (4.3)
cagA−/vacAs1−/vacAm1−
5 (6.5)
5 (7.0)
3 (4.0)
7 (9.6)
6 (7.6)
4 (5.8)
cagA+
62 (80.5)
47 (66.2)
63 (84.0)
46 (63.0)
61 (77.2)
48 (69.6)
cagA−
15 (19.5)
24 (33.8)
12 (16.0)
27 (37.0)
18 (22.8)
21 (30.4)
vacAs1+
64 (83.1)
39 (54.9)
63 (84.0)
40 (54.8)
59 (74.7)
44 (63.8)
vacAs1−
13 (16.9)
32 (45.1)
12 (16.0)
33 (45.2)
20 (25.3)
25 (36.2)
vacAm1+
58 (75.3)
38 (53.5)
61 (81.3)
35 (47.9)
63 (79.7)
33 (47.8)
vacAm1−
19 (24.7)
33 (46.5)
14 (18.7)
38 (52.1)
16 (20.3)
36 (52.2)
Total
77 (100.0)
71 (100.0)
75 (100.0)
73 (100.0)
79 (100.0)
69 (100.0)
↑, upregulated; —, unaltered.*
P
<
0.05 compared with unaltered group. Data are presented as the number of cases and percentage (%).PCR amplification of (a)cagA, (b)vacAs1, and (c)vacAm1 expression inH. pylori extracted from gastric mucosal tissue. Lane M, DNA marker; numbered lanes, samples with positive expression of the target genes. Base pair value indicates fragment of interest.
(a)
(b)
(c)


### 3.3. Correlation betweencagA/vacAs1/vacAm1 andTRAF1/4-1BB/Bcl-xL Expression

To explore the potential association betweencagA/vacAs1/vacAm1 andTRAF1/4-1BB/Bcl-xL expression, we analyzed the number and percentage of samples with positive or negativecagA/vacAs1/vacAm1 expression and differentialTRAF1/4-1BB/Bcl-xLexpression. A remarkably large number of samples with upregulatedTRAF1/4-1BB/Bcl-xL expression werecagA+/vacAs1+/vacAm1+ (44 cases, 67.7%; P
<
0.05; Table 3). From these findings, we believe thatcagA+/vacAs1+/vacAm1+ H. pylori may promote the IM and Ca pathogenesis, possibly by upregulatingTRAF1, 4-1BB,andBcl-xL expression in the gastric mucosal tissue.Table 3
Correlation between expression ofcagA, vacAs1,and vacAm1 and TRAF1, 4-1BB,and Bcl-xL.
Group
T+/4+/B+
T+/4+/B−
T+/4−/B+
T+/4−/B−
T−/4+/B+
T−/4+/B−
T−/4−/B+
T−/4−/B−
Total
cagA+/vacAs1+/vacAm1+
44 (67.7)*
4 (6.2)
1 (1.5)
1 (1.5)
0 (0.0)
2 (3.1)
2 (3.1)
11 (16.9)
65 (100.0)
cagA+/vacAs1+/vacAm1−
2 (12.5)
2 (12.5)
0 (0.0)
2 (12.5)
2 (12.5)
1 (6.3)
0 (0.0)
7 (43.8)
16 (100.0)
cagA+/vacAs1−/vacAm1+
1 (6.7)
0 (0.0)
2 (13.3)
0 (0.0)
2 (13.3)
0 (0.0)
2 (13.3)
8 (53.3)
15 (100.0)
cagA+/vacAs1−/vacAm1−
1 (7.7)
0 (0.0)
1 (7.7)
1 (7.7)
0 (0.0)
2 (15.4)
1 (7.7)
7 (53.8)
13 (100.0)
cagA−/vacAs1+/vacAm1+
2 (22.2)
0 (0.0)
1 (11.1)
0 (0.0)
2 (22.2)
1 (11.1)
0 (0.0)
3 (33.3)
9 (100.0)
cagA−/vacAs1+/vacAm1−
1 (7.7)
0 (0.0)
2 (15.4)
2 (15.4)
0 (0.0)
0 (0.0)
0 (0.0)
8 (61.5)
13 (100.0)
cagA−/vacAs1−/vacAm1+
1 (14.3)
0 (0.0)
0 (0.0)
1 (14.3)
2 (28.6)
0 (0.0)
1 (14.3)
2 (28.6)
7 (100.0)
cagA−/vacAs1−/vacAm1−
3 (30.0)
0 (0.0)
1 (10.0)
1 (10.0)
0 (0.0)
0 (0.0)
2 (20.0)
3 (30.0)
10 (100.0)
*
P
<
0.05 compared with other groups. T, TRAF1; 4, 4-1BB; B, Bcl-xL. Data are presented as the number of cases and percentage (%).


## 3.1.TRAF1,4-1BB, andBcl-xL Expression in Gastric Mucosal Tissue

First, we examinedTRAF1, 4-1BB,andBcl-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,andBcl-xL levels were significantly upregulated in IM and Ca gastric mucosa as compared with CG gastric mucosa (P
<
0.05). The expression of4-1BB was also elevated in AG gastric mucosa compared to CG gastric mucosa (P
<
0.05). Moreover, compared to AG gastric mucosa, there was an obvious increase inTRAF1 protein expression in Ca gastric mucosa (P
<
0.05). These findings show that the expression ofTRAF1, 4-1BB, and Bcl-xL was upregulated, especially in the gastric mucosa of IM and Ca patients in comparison to the other groups. Spearman’s analysis indicated that the expression ofTRAF1, 4-1BB,andBcl-xLwas positively correlated. TheTRAF1and4-1BB, TRAF1andBcl-xL,and4-1BBandBcl-xL correlation coefficient were 0.678, 0.702, and 0.694, respectively (P
<
0.05 for all three groups).Western blotting examination ofTRAF1, 4-1BB,andBcl-xL protein expression in gastric mucosal tissue from subjects with CG, AG, IM, or Ca. (a) Representative western blots. Relative expression of (b)TRAF1, (c)4-1BB, and (d)Bcl-xL was quantified against GAPDH. Data were quantified from three independent experiments. P
<
0.05
* compared with CG; #
P
<
0.05 compared with AG.
(a)
(b)
(c)
(d)


## 3.2. Analysis of Virulence Factors inH. pylori Extracted from Gastric Mucosa

We analyzedH. pylori virulence factors expression,that is, cagA, vacAs1,andvacAm1, inH. pylori extracted from the different gastric mucosal tissues. Representative positive expression of the virulence factors is presented in Figure 2. To clarify the association between theH. pylori virulence factors andTRAF1, 4-1BB,andBcl-xL expression, the number of samples positive or negative forcagA, vacAs1,andvacAm1was stratified according toTRAF1, 4-1BB, andBcl-xL expression (Table 2). According to the results of western blot, the gastric cancer and intestinal metaplasia with atypical hyperplasia groups were classified as theTRAF1, 4-1BB, andBcl-xL high expression group, while the gastritis and atrophic gastritis groups were classified as theTRAF1, 4-1BB, andBcl-xL low expression group. The number and percentage ofcagA-, vacAs1-,andvacAm1-positive cases were greatly upregulated in samples with highTRAF1, 4-1BB,orBcl-xL expression (P
<
0.05 compared with cases negative forcagA, vacAs1, vacAm1). In addition, the number ofcagA+/vacAs1+/vacAm1+ cases in samples with elevatedTRAF1, 4-1BB,orBcl-xLexpression was increased dramatically (P
<
0.05 compared with unalteredTRAF1, 4-1BB,orBcl-xLexpression).Table 2
Samples positive or negative forcagA, vacAs1, or vacAm1 stratified according toTRAF1, 4-1BB,and Bcl-xL expression.
Group
TRAF1
4-1BB
Bcl-xL
↑
—
↑
—
↑
—
cagA+/vacAs1+/vacAm1+
50 (64.9)*
15 (21.1)
50 (66.7)*
15 (20.5)
47 (59.4)*
18 (26.1)
cagA+/vacAs1+/vacAm1−
6 (7.8)
10 (14.1)
7 (9.3)
9 (12.3)
4 (5.1)
12 (17.4)
cagA+/vacAs1−/vacAm1+
3 (3.9)
12 (16.9)
3 (4.0)
12 (16.4)
7 (8.9)
8 (11.6)
cagA+/vacAs1−/vacAm1−
3 (3.9)
10 (14.1)
3 (4.0)
10 (13.7)
3 (3.8)
10 (14.5)
cagA−/vacAs1+/vacAm1+
3 (3.9)
6 (8.5)
5 (6.7)
4 (5.5)
5 (6.3)
4 (5.8)
cagA−/vacAs1+/vacAm1−
5 (6.5)
8 (11.3)
1 (1.3)
12 (16.4)
3 (3.8)
10 (14.5)
cagA−/vacAs1−/vacAm1+
2 (2.6)
5 (7.0)
3 (4.0)
4 (5.5)
4 (5.1)
3 (4.3)
cagA−/vacAs1−/vacAm1−
5 (6.5)
5 (7.0)
3 (4.0)
7 (9.6)
6 (7.6)
4 (5.8)
cagA+
62 (80.5)
47 (66.2)
63 (84.0)
46 (63.0)
61 (77.2)
48 (69.6)
cagA−
15 (19.5)
24 (33.8)
12 (16.0)
27 (37.0)
18 (22.8)
21 (30.4)
vacAs1+
64 (83.1)
39 (54.9)
63 (84.0)
40 (54.8)
59 (74.7)
44 (63.8)
vacAs1−
13 (16.9)
32 (45.1)
12 (16.0)
33 (45.2)
20 (25.3)
25 (36.2)
vacAm1+
58 (75.3)
38 (53.5)
61 (81.3)
35 (47.9)
63 (79.7)
33 (47.8)
vacAm1−
19 (24.7)
33 (46.5)
14 (18.7)
38 (52.1)
16 (20.3)
36 (52.2)
Total
77 (100.0)
71 (100.0)
75 (100.0)
73 (100.0)
79 (100.0)
69 (100.0)
↑, upregulated; —, unaltered.*
P
<
0.05 compared with unaltered group. Data are presented as the number of cases and percentage (%).PCR amplification of (a)cagA, (b)vacAs1, and (c)vacAm1 expression inH. pylori extracted from gastric mucosal tissue. Lane M, DNA marker; numbered lanes, samples with positive expression of the target genes. Base pair value indicates fragment of interest.
(a)
(b)
(c)


## 3.3. Correlation betweencagA/vacAs1/vacAm1 andTRAF1/4-1BB/Bcl-xL Expression

To explore the potential association betweencagA/vacAs1/vacAm1 andTRAF1/4-1BB/Bcl-xL expression, we analyzed the number and percentage of samples with positive or negativecagA/vacAs1/vacAm1 expression and differentialTRAF1/4-1BB/Bcl-xLexpression. A remarkably large number of samples with upregulatedTRAF1/4-1BB/Bcl-xL expression werecagA+/vacAs1+/vacAm1+ (44 cases, 67.7%; P
<
0.05; Table 3). From these findings, we believe thatcagA+/vacAs1+/vacAm1+ H. pylori may promote the IM and Ca pathogenesis, possibly by upregulatingTRAF1, 4-1BB,andBcl-xL expression in the gastric mucosal tissue.Table 3
Correlation between expression ofcagA, vacAs1,and vacAm1 and TRAF1, 4-1BB,and Bcl-xL.
Group
T+/4+/B+
T+/4+/B−
T+/4−/B+
T+/4−/B−
T−/4+/B+
T−/4+/B−
T−/4−/B+
T−/4−/B−
Total
cagA+/vacAs1+/vacAm1+
44 (67.7)*
4 (6.2)
1 (1.5)
1 (1.5)
0 (0.0)
2 (3.1)
2 (3.1)
11 (16.9)
65 (100.0)
cagA+/vacAs1+/vacAm1−
2 (12.5)
2 (12.5)
0 (0.0)
2 (12.5)
2 (12.5)
1 (6.3)
0 (0.0)
7 (43.8)
16 (100.0)
cagA+/vacAs1−/vacAm1+
1 (6.7)
0 (0.0)
2 (13.3)
0 (0.0)
2 (13.3)
0 (0.0)
2 (13.3)
8 (53.3)
15 (100.0)
cagA+/vacAs1−/vacAm1−
1 (7.7)
0 (0.0)
1 (7.7)
1 (7.7)
0 (0.0)
2 (15.4)
1 (7.7)
7 (53.8)
13 (100.0)
cagA−/vacAs1+/vacAm1+
2 (22.2)
0 (0.0)
1 (11.1)
0 (0.0)
2 (22.2)
1 (11.1)
0 (0.0)
3 (33.3)
9 (100.0)
cagA−/vacAs1+/vacAm1−
1 (7.7)
0 (0.0)
2 (15.4)
2 (15.4)
0 (0.0)
0 (0.0)
0 (0.0)
8 (61.5)
13 (100.0)
cagA−/vacAs1−/vacAm1+
1 (14.3)
0 (0.0)
0 (0.0)
1 (14.3)
2 (28.6)
0 (0.0)
1 (14.3)
2 (28.6)
7 (100.0)
cagA−/vacAs1−/vacAm1−
3 (30.0)
0 (0.0)
1 (10.0)
1 (10.0)
0 (0.0)
0 (0.0)
2 (20.0)
3 (30.0)
10 (100.0)
*
P
<
0.05 compared with other groups. T, TRAF1; 4, 4-1BB; B, Bcl-xL. Data are presented as the number of cases and percentage (%).


## 4. Discussion

Previously, we found thatTRAF1, 4-1BB,andBcl-xL were significantly upregulated in GES-1 cells infected withH. pylori expressing the virulence genotypecagA+/vacAs1+/vacAm1+ [8]. Here, we show that expression ofTRAF1, 4-1BB,andBcl-xL was significantly upregulated in IM and Ca patients compared with CG patients. Moreover, compared to samples in whichTRAF1, 4-1BB,orBcl-xL expression was unaltered, there were morecagA+/vacAs1+/vacAm1+ H. pylori–infected cases in samples with elevatedTRAF1, 4-1BB, orBcl-xL expression.TRAF1 belongs to a group of structurally similar adapter proteins (TRAFs), but differs from otherTRAFs 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-κB inducible gene expression [12]. In a previous study, we found thatTRAF1 was upregulated in several gastric cancer cell lines, including BGC823, SGC7901, and MGC803 [13]. Moreover,TRAF1 expression in human gastric mucosa is related to theH. pylori virulence genotypecagA+/vacAs1+/vacAm1+ [9]. In accordance with these findings, we demonstrate here thatTRAF1 was highly expressed in the gastric mucosa of IM and Ca patients and thatTRAF1 upregulation correlated positively with theH. pylori cagA+/vacAs1+/vacAm1+ virulence genotype.Bcl-xL and 4-1BB are two key regulators in theTRAF1 signaling pathway.TRAF1 has a prosurvival effect in CD8 T cells via the4-1BB–mediated upregulation ofBcl-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 inH. pylori–related gastric carcinogenesis [17]. Hence, we investigated4-1BBandBcl-xL expression in the gastric mucosa from different gastric diseases.4-1BB expression was greatly upregulated in AG, IM, and Ca gastric mucosa, andBcl-xL levels were increased, especially in IM and Ca gastric mucosa. Consistent with our findings, Yang et al. reported thatBcl-xL expression was relatively low in CG and AG patients, but was markedly increased in Ca patients [18]. AsTRAF1, 4-1BB, andBcl-xL are correlated positively,TRAF1 may trigger4-1BB–mediatedBcl-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 AsiancagA-positiveH. pylori infection has been reported in gastric cancer patients (84.6%), suggesting thatcagA-positiveH. pylori infection and gastric cancer are closely associated [19]. Furthermore, it has been suggested that thevacAs1+/vacAm1+ genotype is associated with gastric cancer [20]. Importantly, a high proportion of subjects with upregulatedTRAF1, 4-1BB,andBcl-xLexpression in the present study were infected withcagA+/vacAs1+/vacAm1+ H. pylori. In contrast, Matsumoto et al. showed thatH. pylori vacA reducedBcl-xL expression in gastric adenocarcinoma cell lines and led to apoptosis [21]. This discrepancy might be due to the differences betweenin vitro andin 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 thecagA+/vacAs1+/vacAm1+ virulence genotype onTRAF1, 4-1BB,andBcl-xL expression in cultured gastric cancer cells.In summary, our study implies thatcagA+/vacAs1+/vacAm1+ H. pyloriinfection might promote gastritis progression to gastric cancer, possibly by upregulatingTRAF1, 4-1BB,andBcl-xLexpression in gastric mucosal tissue. It is possible thatcagA+/vacAs1+/vacAm1+ H. pylori upregulatesTRAF1 activation, which triggers4-1BB–mediatedBcl-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.



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*Source: 648479-2015-02-08.xml*