A total of 135 patients who underwent surgical resections for primary sporadic gastric carcinoma at the First Affiliated Hospital of Xinxiang Medical University (Weihui, China) between January 2004 and December 2007 were recruited for the study. None of the patients had received preoperative chemotherapy or radiotherapy. In total, 92 males and 43 females (mean age, 58.32 years; range, 28–73 years) were included. The samples obtained included 23 cases of early gastric cancer and 112 cases of advanced gastric cancer, which constituted 31 cases of well-differentiated cancer, 48 cases of moderately differentiated cancer and 56 cases of poorly differentiated cancer. A total of 84 samples were obtained from patients with positive lymph node metastases, while 51 samples were obtained from patients with negative lymph node metastases. All the tumors were adenocarcinomas and were graded according to the World Health Organization criteria (20). Patients were followed-up until mortality or the end of the study (November 30, 2012); the mean postoperative follow-up duration was 50.76±29.70 months. The clinical and pathological characteristics of the samples obtained are shown in Table I. The study was approved by the Ethics Committee of Xinxiang Medical University and written informed consent was provided by the patient’s family,

Genomic DNA was isolated using a DNA extraction kit (Tiangen Biotech Co., Ltd. (Beijing, China). The total DNA content and purity (A₂₆₀/A₂₈₀ >1.8) was measured using an ultraviolet spectrophotometer, and DNA integrity was analyzed using agarose electrophoresis with a gel image analysis system (Syngene, Cambridge, UK). Bisulfite conversion was performed using 1 μg genomic DNA and the CpGenome DNA Modification kit (Intergen, Purchase, NY, USA).

Methylation-specific PCR (MSP) was used to analyze the methylation status of RKIP. RKIP primers were designed in accordance with the study by Al-Mulla et al (21) (Table II). MSP was performed in 25-μl reaction volumes. Following predegeneration at 95°C for 10 min, a total of 40 cycles of 45 sec at 95°C, 30 sec at 53°C and 30 sec at 72°C were completed, followed by a 10 min final extension at 72°C. The PCR products were subsequently observed using 2.0% agarose gel electrophoresis. A water blank was used as a negative control and placental DNA methylated by CpG methyltransferase (M.SssI), in accordance with the manufacturer’s instructions (New England Biolabs, Inc., Beverly, MA, USA), was used as a positive control.

Full tissue sections of 135 paraffin-embedded gastric adenocarcinomas were processed for immunohistochemical staining of RKIP. Specimens were fixed in 4% paraformaldehyde and embedded in paraffin. A 4-μm section from each patient was cut, dried, dewaxed and rehydrated, prior to treatment with 3% hydrogen peroxide solution for 10 min and microwave treatment in citrate buffer (pH 6.0) at 95°C (3×10 min). Nonspecific binding was blocked by treating the slides with 10% normal goat serum for 15 min. The slides were then incubated with rabbit polyclonal antibodies against human RKIP (1:200; Beijing Sequoia Jinqiao Company, Beijing, China) for 20 min at room temperature. Next, the slides were incubated with horseradish peroxidase-labeled streptavidin solution for 20 min at room temperature. Color development was achieved using a 3,3′-diaminobenzidine solution. The slides were counterstained with 1% Meyer’s hematoxylin. As a negative control for RKIP staining, tissue sections were treated with phosphate-buffered saline instead of rabbit polyclonal antibodies against human RKIP. The positive control was a normal gastric mucosa tissue known to express RKIP. Water was used instead of the primary antibody as a negative control.

All statistical analyses were performed using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). The χ² test and two-tailed Fisher’s exact test were used to determine the correlation between RKIP promoter methylation and expression with clinicopathological parameters. The correlation between RKIP promoter methylation and expression was assessed using the Spearman’s rank test. Kaplan-Meier analysis was used to estimate survival as a function of time, and survival differences were analyzed using the log-rank test. Multivariate analysis of the prognostic factors was performed using the Cox proportional hazards model. P<0.05 was considered to indicate a statistically significant difference.

Using the MSP method and immunohistochemical staining, RKIP promoter methylation and expression were detected in 135 pairs of gastric adenocarcinoma tissues and their matched adjacent tissues. The incidence of RKIP promoter methylation in the gastric adenocarcinoma tissues (48.9%) was significantly higher compared with the adjacent mucosa tissues (5.2%; P<0.05). RKIP protein expression was largely localized in the cytoplasm of the normal glands. In a few cells, low levels of RKIP expression were detected in the cell membranes (Fig. 1). The incidence of RKIP protein expression in the tumor tissues (43.0%, 58/135) was significantly lower compared with the adjacent mucosa tissues (91.1%, 123/135; P<0.05; Table III). A total of nine gastric adenocarcinoma cases exhibited positive RKIP expression, while the remaining 57 cases of gastric adenocarcinoma with negative RKIP expression exhibited RKIP promoter methylation. These observations indicated that low RKIP expression was associated with RKIP promoter methylation (P<0.01; Table IV).

Associations between clinicopathological parameters with the methylation status and protein expression of RKIP are presented in Table V. RKIP promoter methylation was shown to significantly correlate with the pathological staging, tumor differentiation, Union for International Cancer Control (UICC) stage and lymph node metastasis (P<0.05). No statistically significant correlations were observed between RKIP promoter methylation and age or gender (P>0.05). RKIP expression was detected in 58.1, 52.1 and 26.8% of cases in the well-differentiated, moderately differentiated and poorly differentiated groups, respectively (P<0.05). RKIP expression was demonstrated to be associated with pathological staging, UICC stage and lymph node metastasis (P<0.05), but no correlations were observed with regard to age or gender (P>0.05).

The five-year overall survival rates of the patients was 43.7%, and the median survival time was 53±5 months. The five-year overall survival rates of patients with RKIP promoter methylation were significantly lower compared with the patients with unmethylated RKIP promoters (P<0.001). Methylation of RKIP was shown to be inversely correlated with the survival of patients with gastric adenocarcinoma (Fig. 2A). The five-year overall survival rates of patients with RKIP-negative tumors were significantly lower compared with those in the RKIP-positive group (P=0.001). RKIP expression positively correlated with the survival rate of gastric adenocarcinoma patients (Fig. 2B). Furthermore, in the multivariate analysis, potential prognosis factors, including lymph node metastasis, UICC-stage and RKIP promoter methylation, were included in the Cox proportional hazards model. The results indicated that in addition to UICC-stage staging and lymph node metastasis, the RKIP promoter methylation status is an independent prognostic factor (P=0.033; Table VI).