Sixty-four chronic hepatitis B patients (CHB) with gastric ulcer were recruited from First Hospital of China Medical University in this study from July 2007 to July 2011. The diagnosis of CHB was confirmed by the serological examination of HBsAg for >6 months. Tissue specimens were derived from the patients after the resection at the Department of General Surgery. This study was in compliance with the Helsinki Declaration, all patients gave written informed consent for participation, and the procedure was approved by Our University Ethics Committee.

Human gastric mucosa cell line, GES-1, was obtained from the American Type Culture Collection (Bethesda, MD, USA) and grown in RPMI-1640 medium (Hyclone, UT, USA) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). Cells were maintained in a humidified cell incubator with 5% CO₂ at 37°C.

A full-length ORF of HBX was obtained from gastric ulcer samples by RT-PCR. The primers of HBX were, sense: 5′-CGGAATTCATGGCTGCTAGGC TGTGCTG-3′ (EcoRI) and antisense: 5′-CGCGGATCCGG CAGAGGTGAAAAAGTTGC-3′ (BamHI) (Takara Dalian, Dalian, China). The cDNA of HBX was cloned into BamHI and EcoRI sites of mammalian expression vector pcDNA3.1. All of the constructs were confirmed by DNA sequencing (Sunbiotech, Beijing, China). Cells were transfected using Lipofectamine™ 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions. HBX-expressing cells were obtained by transfecting with pcDNA3.1-HBX.

Tissues and cells were lysed in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Cell extract protein amounts were quantified using the BCA protein assay kit. Equivalent amounts of protein (20 μg) were separated using 12% SDS-PAGE and transferred to PVDF membrane (Millipore Corporation, Billerica, MA). Western blot was performed using primary antibodies: HBX (Alexis Biochemicals, San Diego, CA), stress-activated protein kinase/JNK antibody (Cell Signaling Technology, Beverly, MA), phospho-stress activated protein kinase/p-JNK (Thr¹⁸³/Tyr¹⁸⁵) (Cell Signaling Technology), CHOP (abcam), BiP (Cell Signaling Technology), c-jun (Cell Signaling Technology), phosphorylated c-jun (Ser63) (Cell Signaling Technology), and β-actin (Millipore). Each specific antibody binding was detected with horseradish peroxidase (HRP)-conjugated, respective, secondary antibodies and ECL solutions (Amersham Biosciences, UK).

Total tissue and cellular RNA was isolated using TRIzol reagent (Invitrogen) and was reverse transcribed by using SuperScript II reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. Real-time PCR was performed using primers specific for HBX and GAPDH. Real-time PCR analysis was performed on the ABI Prism 7500 sequence detection system (Applied Biosystems, Foster, CA) using the SYBR Green PCR Master mixture (Takara, Dalian). The PCR conditions were: one cycle at 95°C for 10 min followed by 40 cycles at 95°C for 15 sec and at 60°C for 1 min. The following primer sets were used: HBX sense: 5′-GGCAGAGGTGAAAAAGTTGC-3′, antisense: 5′-GGCAGAGGTGAAAAAGTTGC-3′; GAPDH sense: 5′-GAA GGTGAAGGTCGGAGT-3′, antisense: 5′-CATGGGTGGAATCATATTGGAA-3′. Relative quantitation was calculated by ΔΔCt method. Each reaction was repeated independently at three times in triplicate.

Immunohistochemistry was used to detect the expression of HBX protein in gastric ulcer samples. The study population included 64 patients. Immunohistochemical staining was performed on 4-μm sections obtained from formalin-fixed, paraffin-embedded blocks. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 min. Antigen retrieval was carried out in citrate buffer (10 mM, pH 6.0) for 30 min at 95°C in a pressure cooker. Anti-HBX (Alexis Biochemicals) at 1:500 was applied incubated at 4°C overnight. Afterward, sections were incubated with a biotinylated secondary antibody and then exposed to a streptavidin complex (HRP). Positive reactions were visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma), followed by counterstaining with hematoxylin. Normal tissue was used as a control. Sections treated without primary antibodies were used as negative controls.

The proliferation rate of HBX-expressing cells and control cells were measured by MTT assay. Briefly, HBX-expressing cells or control cells were plated at a density of 1×10³/well in 96-well plates. After incubation of 48 h, 0.5 mg/ml MTT was added (Sigma). Four hours later, the medium was replaced with 100 μl dimethylsulfoxide (DMSO) (Sigma). Absorbance Optical density (OD) of each well was determined at 490 nm of wavelength with subtraction of baseline reading. Each time point was repeated six times and the mean and standard errors were calculated.

DAPI staining was applied for determining the apoptotic cells. Cells at a density of 1×10⁵ cells/well were maintained on six-well plates and then were treated under the normal culture condition. Cells in each well were individually fixed in 4% (v/v) paraformaldehyde (Sigma) for 15 min and then stained using DAPI (Invitrogen) for apoptotic cells. Cells were then examined and photographed using a fluorescence microscope (Olympus CX71, Japan).

The gastric mucosal GES-1 cells (3×10⁵/well), were plated and incubated overnight. The control and treated cells were trypsinized, collected in PBS and fixed on ice with 1% paraformaldehyde, followed by 70% cold ethanol. After treatment with 10 μg/ml RNase, the cells were stained with 50 μg/ml propidium iodide (PI, KeyGEN, Nanjing, China) for 15 min at room temperature for cell cycle analysis. The apoptotic cells were detected with AnnexinV-FITC/PI double staining. The cells were trypsinized and stained with Annexin V-FITC and PI following the manufacturer’s instructions for the Apoptosis Assay kit (KeyGEN). The stained cells were analyzed by flow cytometry. Data analysis was performed with CellQuest software (BD Biosciences, MD).

All experiments were done three times in triplicate, and the results were expressed as means ± SD (standard deviation). P-values <0.05 were considered to statistically significant. All statistical analyses were performed with SPSS software (version 16.0; SPSS Inc., Chicago, IL, USA).

Western blotting was carried out to investigate the protein status of HBX in gastric ulcer specimens. As shown in the results, the level of HBX protein was higher in gastric ulcers than that in normal tissue (P<0.05, Fig. 1). To examine the relationship between the level of HBX protein and the level of HBX transcription, real-time PCR of HBX mRNA was carried out in gastric ulcer specimens. The results showed that the level of HBX mRNA was also higher than normal tissue and coincident with the level of protein (P<0.05, Fig. 2).

The immunostaining for HBX was only localized in the cytoplasm. HBX protein was highly expressed in gastric ulcer specimens, but not in normal parts of the specimens (Fig. 3). We then analyzed the potential relationship between the expression of HBX and the clinicopathological characteristics of these patients. The results are summarized in Table I. No correlation was found with gender, age, intake of alcohol, ulcer location, or ulcer stage (P>0.05). However, HBx expression was significantly associated with atrophy, metaplasia and bleeding (P<0.05).

In order to study the role that HBX plays in human gastric mucosa cell line GES-1, we examined the effects of exogenous HBX expression in GES-1. To this end, we constructed an HBX-expressing plasmid, pCDNA-3.1-HBX, and transfected it into GES-1 cells. The levels of HBX mRNA and protein increased upon transfection, compared to the levels observed in human gastric mucosa cell line GES-1 (Fig. 5).

MTT assays were performed, and growth inhibition curves were generated (P<0.05, Fig. 4A). Proliferative ratio of the human gastric mucosa GES-1 cells was inhibited by HBX expression. DAPI staining showed morphological changes in the nucleus after HBX transfection. Fig. 4B shows nuclear condensation in apoptotic cells. AnnexinV-FITC and PI double staining was performed to detect apoptotic cells quantitatively. The apoptotic ratio of the cells transfected with pCDNA-3.1-HBX was 6–7 times higher than that of untransfected cells (P<0.05, Fig. 4C). When cell cycles of transfected and untransfected cells were examined using PI staining, the ratio of cells in the G₁ phase increased in transfected cells versus untransfected cells (P<0.05, Fig. 4D).

Given that ER stress is highly correlated with the promotion of apoptosis, in this study we examined the changes of ER-stress mediated apoptotic pathways in GES-1 after pCDNA-3.1-HBX transfection. As shown in Fig. 5, expression of ER stress molecules (BiP and CHOP) was significantly increased in HBX-transfected cells. Activation of JNK pathway in GES-1 cells transfected with pCDNA-3.1-HBX was confirmed using a phosphorylated JNK-specific antibody and by detecting the phosphorylation of c-jun (Fig. 5). No significant changes of JNK and c-jun were detected in transfected cells. These results indicate that JNK phosphorylation is a crucial event controlling HBX-induced apoptosis. We also confirmed that caspase-3 was activated in transfected cells.