The GC cell lines MKN1, MKN45, MKN74, NUGC2, NUGC3, NUGC4 and SC-6-JCK were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). The AGS, KATOIII and N87 cell lines were acquired from the American Type Culture Collection (Manassas, VA, USA). The GCIY was obtained from Tohoku University, Sendai, Japan. A control, non-tumorigenic epithelial cell line (FHs 74) was purchased from the American Type Culture Collection. Cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) and maintained in a 5% CO₂ atmosphere at 37°C. For FHs 74 cells, the medium was additionally supplemented with 30 ng/ml epidermal growth factor (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA). Total RNA was extracted using an RNeasy Mini kit (Qiagen GmbH, Hilden, Germany) and used as a template for the generation of complementary DNA as described previously (16,17). Primary GC tissues and corresponding normal adjacent tissues were collected from 175 patients who underwent gastric resection for GC without neoadjuvant therapy at Nagoya University Hospital (Nagoya, Japan) between November 2001 and December 2012. Patients who received neoadjuvant therapy were excluded, as it was difficult to obtain cancer cells from scarred tissues. Following collection, tissue samples were immediately frozen in liquid nitrogen and stored at −80°C until the time of RNA extraction. Corresponding normal adjacent gastric mucosa samples were obtained from each patient and were collected from a region no less than 5 cm from the tumor edge. To determine whether the expression status of SAMSN1 differed according to tumor histology, patients were categorized into two histological subtypes: Differentiated (papillary, well differentiated and moderately differentiated adenocarcinoma) and undifferentiated (poorly differentiated adenocarcinoma, signet ring cell carcinoma and mucinous carcinoma) (18). Since 2006, adjuvant chemotherapy using S-1 (an oral fluorinated pyrimidine) has been administered to all Union for International Cancer Control (UICC) stage II/III GC patients (unless contraindicated by the patient's condition) (19,20). Patients were followed-up at least once every 3 months for 2 years following surgery, and then every 6 months for 5 years or until death. Physical examination, laboratory tests and enhanced computed tomography (chest and abdominal cavity) were performed at each visit (21). The chemotherapy regimen for patients with distant metastasis or recurrence was chosen at the physician's discretion. The present study conformed to the ethical guidelines of the World Medical Association Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Subjects, and was approved by the Institutional Review Board of Nagoya University, Nagoya, Japan. Written informed consent for usage of clinical samples and data, as required by the institutional review board, was obtained from all patients (22).

SAMSN1 mRNA expression levels in 11 GC cell lines and 175 primary GC tissues and corresponding normal adjacent tissues were analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using an ABI StepOnePlus Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) in conjunction with the gene specific primers listed in Table I. Cycling conditions were as follows: One cycle at 95°C for 10 min, 40 cycles at 95°C for 5 sec and 60°C for 60 sec To investigate the oncological role of SAMSN1 in GC, correlation analysis was performed to evaluate the association between the pattern of SAMSN1 mRNA expression and clinicopathological parameters, including patient survival following gastrectomy. Each of the 175 patients was assigned to one of two groups (low and high SAMSN1 expression) according to their median level of SAMSN1 mRNA expression in GC tissues. Additionally, the prognostic impact of SAMSN1 mRNA expression on patients categorized according to the 7th UICC staging system was also evaluated (23).

Genomic DNA from GC cell lines was treated with bisulfite using the EpiTect Bisulfate kits (Qiagen GmbH) and sequenced to determine the levels of DNA methylation according to previously published procedures (24).

The intensity and pattern of SAMSN1 protein expression was determined by immunohistochemical staining using 48 representative sections of well-preserved GC tissue as described previously (25). Sections were incubated for 1 h at room temperature with a rabbit polyclonal antibody raised against SAMSN1 (catalog no., 13063-1-AP; ProteinTech Group, Inc., Chicago, IL, USA) diluted 1:400 in antibody diluent (Dako, Glostrup, Denmark). The samples were subsequently washed with phosphate buffered saline, followed by a 10 min incubation with biotinylated rabbit secondary antibody (Histofine SAB PO(R) kit; Nichirei Corporation, Tokyo, Japan) in a 1:1,000 dilution with ChemMateT antibody diluent (Dako). Sections were subsequently developed for 3 min using 3,3′- diaminobenzidine as the substrate (Nichirei Corporation). The patterns of SAMSN1 staining in GC tissues and corresponding non-cancerous tissues were compared, and positive blood vessel staining provided an internal control for the immunolabeling procedure. Specimens were randomized and coded prior to analysis by two independent observers blinded to the status of the samples (26,27).

Differences in the relative expression of SAMSN1 mRNA (normalized to the level of glyceraldehyde-3-phosphate expression) between the two groups were analyzed using the Mann-Whitney U test. The χ² test was used to analyze the association between the expression status of SAMSN1 and various clinicopathological parameters. A correlation between expression patterns of SAMSN1 protein and mRNA in gastric tissue specimens was also evaluated by the χ² test. Survival rates were calculated using the Kaplan-Meier method, and the difference in survival curves was analyzed using the log-rank test. Multivariate regression analysis was performed to detect prognostic factors using the Cox proportional hazards model, and variables with P<0.05 were entered into the final model. All statistical analysis was performed using JMP version 10 software (SAS Institute Inc., Cary, NC, USA). P<0.05 was considered to indicate a statistically significant difference.

A marked decrease in the level of SAMSN1 mRNA expression was detected in 8 (73%) of the 11 GC cell lines when compared with the FHs 74 control cell line. There was no marked difference in SAMSN1 expression between cell lines derived from differentiated and undifferentiated GCs (Fig. 1A). No DNA methylation of the SAMSN1 promoter was detected.

The patient population included 134 males and 41 females with an age range from 20–84 years (mean age, 64.7±11.8 years). Pathologically, 106 patients were diagnosed with undifferentiated GC and 69 with differentiated GC. A total of 39 patients were diagnosed with stage I disease, 29 with stage II, 51 with stage III and 56 with stage IV disease. A total of 119 patients with stage I–III disease underwent R0 resection. A total of 47/56 patients classified as UICC stage IV were assigned this diagnosis due to positive peritoneal lavage cytology, localized peritoneal metastasis or distant lymph node metastasis. A total of 6 of the patients with stage IV disease had synchronous liver metastasis and a single patient had lung metastasis, and these individuals underwent gastrectomy to control bleeding or obstruction to the passage of food.

The mean expression level of SAMSN1 mRNA was reduced in GC tissues when compared with that in adjacent normal tissues (P<0.001). However, there was no significant difference in the expression of SAMSN1 mRNA between patients with undifferentiated and differentiated GC (Fig. 1B; P=0.067). Immunohistochemical staining was subsequently performed to investigate the expression of SAMSN1 protein in those cases where the SAMSN1 mRNA level in GC tissues was observed to be less or equivalent to that identified for corresponding non-cancerous tissues. Representative GC specimens with an increased, equivalent and reduced intensity of SAMSN1 protein staining in cancerous tissue compared with adjacent normal tissue are shown in Fig. 2A. In 48 of the patient samples examined, the pattern of SAMSN1 protein expression correlated significantly with that of the expression of SAMSN1 mRNA (P=0.005; Fig. 2B).

Patients were assigned to one of two groups according to their median SAMSN1 mRNA expression level in GC tissues (high expression group, n=87; low expression group, n=88). Low SAMSN1 mRNA expression was significantly associated with larger tumor size (>60 mm; P=0.026), but not tumor location or UICC stage (P=0.639) (Table II). Patients in the low SAMSN1 expression group were more likely to have a shorter overall survival time than those in the high expression group (5-year survival rates were 43% and 66% for the high and low expression groups, respectively; P=0.004; Fig. 3A). In multivariate analysis for overall survival, low SAMSN1 mRNA expression was identified to be an independent prognostic factor (hazard ratio, 1.80; 95% confidence interval, 1.07–3.05; P=0.025; Table III). When patients were categorized according to UICC stage, no significant differences in the mean expression level of SAMSN1 mRNA was observed between groups (P>0.05, for each), suggesting that SAMSN1 expression was independent of tumor stage (Fig. 3B).

Subsequently, a subgroup analysis of patients categorized according to UICC stage was performed. The survival difference between the low and high SAMSN1 expression groups was more apparent in patients with stage II/III GC (P=0.025_ than those with stage IV GC (P=0.162) (Fig. 4A). Among 80 patients with stage II/III GC who underwent curative surgery, those who had a low level of SAMSN1 mRNA expression in GC tissues were more likely to have shorter disease free survival times than those who had high SAMSN1 mRNA expression (2-year survival rates were 50% and 81% for the low and high SAMSN1 expression groups, respectively; P=0.038; Fig. 4B).