Diaminobenzidine (DAB) was obtained from Golden Bridge International (Beijing, China) and Dulbecco's modified Eagle's medium (DMEM) was obtained from Life Technologies (New York, NY, USA). Fetal bovine serum (FBS), Lipofectamine 2000, TRIzol reagent and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were provided by Invitrogen (Carlsbad, CA, USA). The SuperScript II reverse transcriptase kit was purchased from Takara Bio Inc. (Otsu, Shiga, Japan) and the enhanced chemiluminescence (ECL) reagent and polyvinylidene fluoride (PVDF) membranes were from Merck Millipore (Darmstadt, Germany). The RIPA lysis buffer kit was from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the protein assay kit was purchased from Bio-Rad (Hercules, CA, USA). Cobalt dichloride (CoCl₂) and other reagents were from Sigma (St. Louis, MO, USA) unless otherwise stated.

Tissue specimens from 178 GC patients were collected from consecutive surgical cases at the Department of Surgical Oncology, The First Affiliated Hospital, Medical School, Xi'an Jiaotong University and the Department of Surgical Oncology, The 215th Hospital of Shaanxi Province, between 2004 and 2009. The patients included 125 males and 53 females, ranging between the ages of 25 and 81 years. Histological diagnosis of GC was assessed according to the criteria of staging of the primary tumor/regional lymph nodes/distant metastasis (TNM) as described in the AJCC Cancer Staging Manual (25). The clinicopathological data of the patients are documented in Table II. In brief, tumors penetrating into the serosa or those involved in adjacent structures were observed in 77 patients (43.25%), 129 patients (72.47%) had lymph node metastases and 40 patients (22.47%) had distant metastasis. Twenty-six patients underwent total gastrectomy, whereas the remaining 152 patients underwent partial resection of tumor lesions. Among them, 178 patients did not receive any neoadjuvant or adjuvant chemotherapy before surgery, and 108 of 178 patients received fluorouracil-based postoperative adjuvant chemotherapy. The present study was approved by the Protection of Human Subjects Committee of The First Affiliated Hospital, Medical School, Xi'an Jiaotong University and complies with the Helsinki Declaration.

Tissue specimens were fixed in a buffered formalin solution and embedded in paraffin for serial sections (4-µm in thickness) that were mounted onto charged glass slides. For immunohistochemistry, the tissue sections were subjected to deparaffinization and rehydration and then to antigen retrieval in a citrate buffer at pH 6.0 in a microwave. The sections were then subsequently incubated with a goat polyclonal antibody against RAP1B (sc-1481; Santa Cruz Biotechnology) at a dilution of 1:600 or anti-HIF-1α (bs0737R; Biosynthesis Biotechnology, Beijing, China) at a dilution of 1:500 overnight at 4°C. The streptavidin-peroxidase technique (SP-9001 and PV-9003; Golden Bridge International) was used to amplify the signal, and the sections were incubated with 0.02% DAB solution to develop the positive color reaction, followed by brief counterstaining with hematoxylin. Some duplicated sections were also incubated with an irrelevant rabbit antiserum to replace the first antibody which served as a negative control.

The immunostained tissue sections were reviewed and scored under a microscope independently by two experienced pathologists, who were blinded to the clinical data with consensus. The staining results for RAP1B and HIF-1α proteins were scored semi-quantitatively by addition of the immunostaining intensity and the percentage of positive tumor cells. The percentage of positive malignant cells was determined in at least five fields under a ×400 magnification and was averaged as follows: no staining or <5%, 0 points; between 5 and 25%, 1 point; between 26 and 50%, 2 points; between 51 and 75%, 3 points; and >75%, 4 points. The staining intensity was scored as follows: no coloring, 0 points; slightly yellow, 1 point; brown yellow, 2 points; and tan stains, 3 points. If the sum of these two scores was ≤5, the section was defined as having low expression; whereas ≥6 was defined as high expression.

The human GC cell lines MKN28, BGC823 and SGC7901 were obtained from the Cell Bank of Shanghai (Shanghai, China) and cultured in DMEM with high glucose and supplemented with 10% heat-inactivated FBS at 37°C in a humidified incubator under an atmosphere of 5% carbon dioxide. All experiments were performed with cells in the logarithmic phase of growth.

Cancerous cells were seeded into 96-well plates at a density of 10⁴ cells/well and incubated at 37°C for 24 h. Next, fresh growth medium containing the various concentrations of CoCl₂ (at concentrations of 50–300 µM) was added into each well. Following incubation at 37°C for 24 h, the cell viability was assessed using the MTT colorimetric assay. All experiments were conducted in triplicate and repeated at least three times.

To assess the alteration of tumor cell invasion capacity, a Transwell assay was performed using a membrane invasion culture system (Corning, Corning, NY, USA), following the standard protocol. Briefly, control or siRAP1B-transfected GC cells (3–5×10⁴/well) were resuspended in 200 µl of serum-free DMEM with high glucose containing 150 or 200 µM CoCl₂ and were added into the upper compartment of the Transwell chambers. A total of 600 µl of DMEM with high glucose containing 20% (v/v) FBS and 150 or 200 µM CoCl₂ was added into the bottom chamber as a source of chemoattractant. After incubation at 37°C in a 5% CO₂ incubator for 24 h, the non-invaded tumor cells on the upper surface of the membrane were removed with a cotton swab, and the tumor cells that invaded into the lower surface of the membrane were fixed with 4% paraformaldehyde, stained with 1% crystal purple, reviewed and randomly counted in five visual fields of each membrane at a magnification of ×200 under a light microscope. Each experiment was performed in triplicate and repeated once.

Small interfering RNA (siRNA) molecules targeting human RAP1B were designed and synthesized by GenePharma Co. (Shanghai, China). The target sequences were as follows: siRAP1B-439, 5′-GAG GGA UUU AUA CAU GAA ATT-3′ and 5′-UUU CAU GUA UAA AUC CCU CTT-3′; siRAP1B-501, 5′-CCA CAU UUA ACG AUU UAC ATT-3′ and 5′-UGU AAA UCG UUA AAU GUG GT-3′; siRAP1B-722, 5′-GUG CGG CAA AUU AAC AGA ATT-3′ and 5′-UUC UGU UAA UUU GCC GCA CTT-3′; and negative control siRNA, 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and 5′-ACG UGA CAC GUU CGG AGA ATT-3′. The GC cells were grown and transfected with the negative control or RAP1B siRNAs using Lipofectamine 2000 according to the manufacturer's protocol. After transfection for 48 h, the cells were harvested for RNA isolation and western blot analysis of RAP1B expression.

Total cellular RNA was isolated using a TRIzol reagent and reversely transcribed into cDNA using the SuperScript II reverse transcriptase kit, according to the manufacturer's instructions. The synthesized cDNA was then subjected to qPCR amplification using primers: [RAP1B, 5′-TTT ATT CCA TCA CAG CAC AGT CC-3′ and 5′-TTT CTG TTA ATT TGC CGC ACT AGG-3′; glyceralde-hydes-3-phosphate dehydrogenase (GAPDH), 5′-CTG GGG CTC ATT TG-3′ and 5′-CAT CCA CAG TCT TC-3′] under the following conditions: 95°C for 30 sec and 30 cycles of 95°C for 5 sec, 60°C for 30 sec and 72°C for 60 sec for 30 cycles. The quantity of each transcript was normalized to that of GAPDH.

Whole-cell lysates were prepared from cell lines using the RIPA lysis buffer kit, and the protein concentrations were quantified using the Bio-Rad protein assay. Equal amounts of protein samples were separated onto a 10 or 12% sodium dodecyl sulfate-polyacryl-amide gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred onto a PVDF membrane. For western blotting, the membranes were probed with antibodies against RAP1B and HIF-1α. Specifically, the membranes were blocked in tris-buffered saline with Tween-20 containing 5% non-fat dry milk and were then incubated overnight with a primary antibody, followed by horseradish peroxidase-conjugated secondary antibodies at room temperature. The blots were visualized with the ECL detection system. β-actin was used as an internal control and the blots were analyzed densitometrically using Quantity One software (Bio-Rad).

Statistical analysis was performed using the SPSS software package version 16.0 (SPSS, Inc., Chicago, IL, USA). The Chi-square test was used to analyze the association of RAP1B and HIF-1α expression with the clinicopathological variables. The Spearman's rank correlation coefficient test was performed to associate HIF-1α expression with RAP1B expression in tissue specimens. Overall survival was defined as the time from the date of surgery to the date of last follow-up or death of the patients. Survival curves were calculated using the Kaplan-Meier method and compared using the log-rank test. Multivariate analysis using Cox's proportional hazard model was used to assess prognostic factors. For each treatment and control, data from the independent replicate trials were pooled, and the results were expressed as the means ± standard error. All continuous data were tested for normality with the Kolmogorov-Smirnov test. The Student's t-test or one-way analysis of variance was used to compare normally distributed variables. A P-value of ≤0.05 was considered to indicate a statistically significant result.

In the present study, we first assessed the protein expression of RAP1B and HIF-1α using immunohistochemistry in 178 GC tissue specimens (Fig. 1A as a negative control) and found that RAP1B protein was localized in the nuclei or cytosol (Fig. 1B), while HIF-1α protein was predominantly in the nuclei; concomitant cytoplasmic staining was ignored since HIF-1α protein is functionally active in the nuclei (Fig. 1C). Expression of these two proteins was significantly different between the cancer and para-cancer normal tissues (Table I). Expression of RAP1B protein was significantly associated with tumor size (diameter), depth of invasion, regional lymph nodes, distant metastasis and TNM staging, yet not with gender, age, tumor differentiation or Borrmann type (Table II). Moreover, HIF-1α expression was associated with these clinicopathological variables, except for gender, age and tumor diameter. In addition, RAP1B expression was associated with HIF-1α expression in the GC tissues. Specifically, Spearman's rank correlation coefficient test showed that there were 98 cancer cases expressing a high level of RAP1B vs. 96 cases with HIF-1α expression (Table III), revealing a positive association (r=0.547, P<0.001).

The Kaplan-Meier curves and the log-rank test data showed that patients with a high RAP1B-expressing tumor had a statistically significant poor overall survival compared to the patients with a low RAP1B-expressing tumor (Fig. 2A and Table IV); 30 months [95% confidence interval (CI), 27.01–32.98] vs. 49 months (95% CI, 41.20–56.79; P<0.001). The same was true for HIF-1α expression (Fig. 2B and Table IV). Furthermore, TNM stage and tumor size (diameter) were all associated with a poor overall survival of patients (P<0.05). The multivariate Cox regression model showed that RAP1B expression [P<0.001, relative risk (RR)=2.329, 95% CI, 1.600–3.390] and TNM stage (P<0.001, RR=1.708, 95% CI, 1.318–2.112) were independent prognostic indicators for survival of GC patients (Table V).

Our ex vivo data demonstrated that RAP1B and HIF-1α protein expression was associated with malignant behaviors of GC and a poor overall survival; thus, we further assessed the role of hypoxia in the regulation of GC phenotypes and gene expression by treating tumor cells with CoCl₂ to mimic in vivo hypoxic conditions. The cell viability MTT assay assessed the proper concentrations of CoCl₂ in vitro (Fig. 3). Next, we utilized the Transwell tumor cell invasion assay to detect the invasion capacity of MKN28, BGC823 and SGC7901 cells and found that the numbers of invaded tumor cells were increased following treatment with CoCl₂ (P<0.05; Fig. 4). These results suggest that hypoxia increases the invasion capacity of GC cells.

In order to verify whether hypoxia regulates RAP1B expression and induces GC cell invasion, we treated MKN28, BGC823 and SGC7901 cells with 150 or 200 µM CoCl₂ for 24 h and found that the expression of both RAP1B mRNA and protein was upregulated (P<0.05; Fig. 5); similarly, HIF-1α was upregulated at the protein level (P<0.05; Fig. 5B). Thus, hypoxia induced RAP1B and HIF-1α expression in GC cells.

To illuminate RAP1B function in GC cells, we knocked down RAP1B using siRNAs and found that siRAP1B-501 was shown to have the best efficiency; thus, it was used for the next experiments (Fig. 3D). We then transfected negative control siRNA and siRAP1B-501 into GC cells, treated the cells with 150 or 200 µM CoCl₂ for 24 h, and performed the Transwell tumor cell invasion assay. Our data showed that MKN28, BGC823 and SGC7901 cells all demonstrated reduced invasion capacity, even after CoCl₂ treatment (Fig. 6), compared to the negative control siRNA-transfected cells after treatment with CoCl₂ (P<0.001). However, knockdown of RAP1B expression did not repress HIF-1α expression under hypoxic conditions (Fig. 7). Hence, RAP1B plays an important role in GC invasion under a hypoxic environment.