A total of 105 patients with gastric cancer undergoing radical gastrectomy at The First Affiliated Hospital of Henan University of Science and Technology (Henan, China) were recruited in the current study between October 2013 and October 2015. There were 63 males and 42 females, with mean age of 53.7±12.8 years old (range, 39–75 years). Gastric cancer tissue and normal gastric tissue adjacent to the carcinoma were taken from all patients during radical resection of gastric tumors. Lesions were not detected in normal adjacent tissues following pathological examination. The tumor location in each patient varied. There were 46 cases in the gastric antrum/pylorus, 36 cases in gastric fundus/cardia and 23 cases in the gastric body. Additionally, the tumor-node-metastasis (TNM) stages of all patients were assessed (13). There were 46 patients with TNM stage II and 59 patients had stage III. The current study was conducted in accordance with the declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Henan University of Science and Technology (Henan, China). Written informed consent was obtained from all participants.

A total of 0.1 g tissue was weighed and TRIzol (Takara, Biotechnology, Co., Ltd., Dalian, China) was added. The tissue was cut into small pieces using surgical scissors and ground using a glass homogenizer. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. The final precipitation was dissolved in RNase free water, the optical density (OD) value of total RNA and OD₂₆₀/OD₂₈₀ were recorded to calculate RNA purity. RNA was transcribed into cDNA using the PrimeScript RT Reagent kit (Takara Biotechnology, Co., Ltd.), following the manufacturer's instructions. GAPDH was used as a reference gene. The primers used were as follows: ERO1L, forward, 5′-CCATTAGTGCTGCCA-ACCAGTA-3′ and reverse, 5′-ATCTGCATCAGCATCACGGTC-3′; GAPDH, forward, 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse, 5′-AGGGGCCATCCACAGTCTTC-3′. The primer was diluted to 10 µmol/l⁻¹. A PCR reaction system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was prepared with a total reaction volume of 20 µl. The PCR reaction conditions were as follows: Initiation for 3 min at 94°C, degeneration for 30 sec at 94°C, annealing for 30 sec at 60°C and extension for 30 sec at 72°C, for a total of 35 cycles. The data were directly read from the ABI 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and quantitatively analyzed using the 2^−ΔΔCq method (14).

A total of 0.15 g gastric cancer and normal tissue was weighed and cut into pieces using the scissors. The sample was ground using the glass homogenizer, washed using 10X pre-cooled PBS, centrifuged at 3,000 × g for 5 min at 4°C. The supernatant was removed, radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) was placed in the ice box, lyzed for 30 min and centrifuged at 15,000 × g for 10 min at 4°C. The supernatant was removed and protein concentration was determined using the BCA protein assay kit (Beyotime Institute of Biotechnology), 5X SDS sample buffer was added; the final concentration was 1X. The sample was boiled for 30 min prior to 12% SDS-PAGE (100 ng protein per lane). The protein was then transferred onto PVEF membranes and blocked with 5% nonfat milk powder for 30 min at room temperature. Mouse anti human ERO1L monoclonal antibody (dilution, 1:1,000; cat. no. ab57177; Abcam, Cambridge, UK) was added, incubated overnight at 4°C, prior to washing with PBST three times for 5 min each time. Goat-anti-mouse immunoglobulin G monoclonal antibody (dilution, 1:2,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was added and incubated at room temperature for 1 h. The 3,3′-diaminobenzidine (DAB) reagent (Beyotime Institute of Biotechnology) was used for visualization and photographed. GAPDH antibody (dilution, 1:2,000; cat. no, sc-25778; Santa Cruz Biotechnology, Inc.) was used as an internal reference, and incubated overnight at 4°C. Results were analyzed using the Image J software (version 1.38, National Institutes of Health, Bethesda, MD, USA) and the relative expression of target protein was calculated.

The tissue specimens, including controls and gastric cancer tissue, were embedded in paraffin using embedding agent at room temperature for 1 h, continuously cut into 4 mm slices, adhered on a glass slide processed with polylysine, baked in an oven at 50°C for 1 h, hydrated using xylene and 100, 95, 80 and 75% ethanol and washed three times with distilled water. The slides were then arranged in the buffer solution containing sodium citrate, heated twice for 8 min each time and washed with PBS three times. Slides were then incubated in 3% H₂O₂ solution at room temperature for 10 min to block endogenous catalase activity and followed by three washes with PBS 5 min each time. The ERO1L antibody (dilution, 1:100; cat. no. ab57177) was added, incubated overnight at 4°C and washed 3 times using PBST for 5 min each time. Subsequently, 10% goat-anti-mouse secondary antibody (dilution, 1:2,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) was added, incubated at room temperature for 1 h and washed for 3 times using PBST for 5 min each time. DAB development, staining with hematoxylin, 0.1% hydrochloric acid differentiation, dehydration, transparency and neutral resin sealing were performed successively. A fluorescent microscope (BX61 Automated Fluorescent Microscope; Olympus Corporation, Tokyo, Japan) was used to observe the above results at magnification, ×200.

Microscope observation demonstrated that ERO1L protein was primarily detected in the cytoplasm. The staining was pale yellow and brown and cells were analyzed according to cell staining intensity. For each tissue section, 5 high power fields were selected. A total of 100 target cells were counted under each high power field. The average percentage of 5 fields was judged as the result. Staining evaluation was performed as follows: 0, all negative; 1, positive cells ≤10%; 2, positive cells 11–50%; 3, positive cells 51–75%; 4, positive cells >75%. Staining intensity was evaluated as follows: 0, no staining; 1, pale yellow; 2, brown; 3, deep yellow. The two scores were multiplied and the total score was the demarcation point. <4 was determined to indicate negative ERO1L expression and ≥4 was determined to indicate positive expression of ERO1L. The cumulative survival and recurrence rates were evaluated in the ERO1L positive and negative groups.

All data were analyzed using SPSS 17.0 statistical software (SPSS, Inc, Chicago, IL, USA). All measurement data are presented as the mean ± standard deviation. The measurement data among groups were compared using the Student's t-test. The cumulative survival and recurrence rates were analyzed using the log-Rank test. P<0.05 indicated a statistically significant difference.

The results of RT-qPCR indicated that ERO1L mRNA levels in gastric cancer tissues were significantly higher compared with normal adjacent tissues and that this difference was statistically significant (P<0.05; Fig. 1).

The results of the western blot analysis demonstrated that, compared with adjacent tissues, ERO1L expression in gastric cancer tissue was significantly higher (P<0.05; Fig. 2).

As presented in Fig. 3, the results of immunohistochemistry demonstrated that ERO1L was primarily expressed in the cytoplasm, but was not expressed in the nucleus or cell membrane. Results from immunohistochemistry demonstrated that expression of ERO1L was negative in the adjacent tissues, whereas its expression was markedly increased in gastric cancer tissues. In gastric cancer tissue, the expression of ERO1L was positive in 83 cases and negative in 22 cases.

As presented in Fig. 4, all patients were followed up for >36 months. Kaplan-Meier curve indicated that the cumulative recurrence rates at 1, 2 and 3 years were 25.30% (n=21), 38.55% (n=32) and 48.19% (n=40) in ERO1L positive patients; The cumulative recurrence rates within 1, 2 and 3 years were 13.64% (n=3), 22.73% (n=5) and 31.82% (n=7) respectively in ERO1L negative patients. The cumulative recurrence rate of ERO1L positive patients was significantly higher than that of negative patients, and the difference was statistically significant (P<0.05; Fig. 4A). Log-Rank test indicated that the cumulative survival rates for 1, 2 and 3 years were 75.90% (n=63), 63.86% (n=53) and 49.40% (n=41) respectively in patients positive for ERO1L. By contrast, the cumulative survival rates for 1, 2 and 3 years were 90.91% (n=20), 81.82% (n=18) and 72.73% (n=16) respectively, in patients negative for ERO1L. The cumulative survival rate in patients positive for ERO1L was significantly lower than that of patients negative for ERO1L (P<0.05; Fig. 4B).