Approval for the present study was obtained from the Ethics Committee of The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China and informed consent was obtained from all patients prior to enrollment in this study. Gastric cancer tissues, tumor-adjacent tissues and normal gastric tissues were surgically resected from 32 patients with primary gastric cancer. None of the 32 patients had receivedchemotherapy or radiotherapy prior to surgery. The specimens were immediately used for mRNA and protein extraction.

AGS cells purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (FBS) (both from Invitrogen, Carlsbad, CA, USA) in the presence of 5% CO₂ at 37°C. The culture medium was replaced every 2–3 days. EGF was purchased from Sigma (St. Louis, MO, USA) and dissolved in PBS at different concentrations (5, 10, 25, 50 and 100 ng/ml) for cell stimulation.

Total RNA was extracted using TRIzol reagent (Invitrogen). Total RNA (2 μg) was used to perform reverse transcription using the RevertAid First Strand cDNA Synthesis kit (Fermentas, Vilnius, Lithuania). Real-time PCR was performed on a 7900 real-time PCR system using the SYBR-Green PCR kit (both from Applied Biosystems, Foster City, CA, USA). All procedures were carried out according to the manufacturer’s instructions. The Ct value was calculated using the ΔΔCt method.

The protein samples from the tissues and cultured cells were isolated using a total protein extraction kit (Sangon Biotech, Shanghai, China) following the manufacturer’s instructions. Protein concentrations were examined using the BCA method with reagents from Pierce (Rockford, IL, USA). Protein samples were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes. Subsequently, the membranes were incubated in blocking solution (Tris-buffered saline containing 5% non-fat milk and 0.05% Tween-20) at 4°C overnight. The blots were probed with the primary antibodies [rabbit polyclonal to CO-029 (sc-292058), 1:400 dilution; rabbit polyclonal to β-actin (sc-130656), 1:400 dilution] followed by IgG-horse radish peroxidase (HRP)-conjugated secondary antibody [goat anti-rabbit IgG-HRP (sc-2004), 1:2,000 dilution] (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The signal was detected using an ECL western blotting kit (Pierce) and the LabWork 4.0 software (UVP Inc., Upland, CA, USA) was used for semi-quantitative analysis. β-actin was used as a loading control.

For siRNA transfection, the cells were seeded into 6-well plates and allowed to grow to approximately 70% confluence. The cells were then transfected with 2 μM CO-029 siRNA or scramble control siRNA using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Following incubation with the transfection mixtures for 4 h, the medium was removed and the cells were incubated with fresh Dulbecco’s modified Eagle’s medium containing 10% FBS for 24 h. Scramble control siRNA (Scr)-transfected cells were incubated with PBS or EGF; CO-029 siRNA (Si)-transfected cells were incubated with EGF.

Cell proliferation was determined by MTT assay. The cells were seeded into 96-well plates and EGF was added to the medium. Following treatment for 12, 24, 48 and 72 h, the cells were incubated with 10 μl MTT (Sigma) at 37°C for 4 h. The medium was then removed and the cells were incubated with 200 μl dimethyl sulfoxide to solubilize the formazan crystals. Untreated cells were used as the contrls. The absorbance at 570 nm was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Cells in the blank group were incubated with Dulbecco’s modified Eagle’s medium containing 10% FBS. PBS was added to the control group as the vehicle control. Cell invasion ability was evaluated using Transwell inserts coated with Matrigel. Transwell inserts (Corning, Inc., Corning, NY, USA) were coated with Matrigel matrix (BD Biosciences, Franklin Lakes, NJ, USA) at a final concentration of 200 μg/ml. The cell plates with the coated inserts were incubated at 37°C for 2 h. Cell suspension was prepared in serum-free medium containing 5×10⁴ cells/ml and added to the upper chambers. A total of 1 ml of cell medium containing 10% FBS was added to the lower chambers. The cell invasion chambers were incubated overnight in a 37°C, 5% CO₂ atmosphere. A cotton swab was used to gently remove the non-invaded cells. The cells on the lower surface of the membrane were fixed in 95% ethanol and then stained with hematoxylin for 10 min. The number of invaded cells was evaluated by counting the cells under an inverted microscope (Nikon, Tokyo, Japan).

Descriptive data are presented as the means ± standard deviation (SD). Differences between 2 groups were evaluated using the Student’s t-test. A P-value ≤0.05 was considered to indicate a statistically significant difference.

The expression of CO-029 in human normal gastric tissues, gastric cancer tissues and tumor-adjacent tissues was analyzed by RT-qPCR and western blot analysis. The mRNA expression of CO-029 did not differ significantly between the normal gastric tissues and the tumor-adjacent tissues (P>0.05); however, the CO-029 mRNA expression was significantly increased in the gastric cancer tissues compared with both the normal gastric tissues and the tumor-adjacent tissues (P<0.01; Fig. 1). The results from western blot analysis revealed that CO-029 protein expression was detectable in the human normal gastric tissues, gastric cancer tissues and tumor-adjacent tissues. Similar to the results obtained for the mRNA expression, the gastric cancer tissues showed an upregulated protein expression of CO-029 in comparison to the normal gastric tissues and tumor-adjacent tissues (P<0.05l; Fig. 2).

The cells were treated with various concentrations of EGF for 24 h, and western blot analysis was then performed to examine the effect of EGF on CO-029 protein expression. We observed that treatment with EGF increased the expression level of CO-029 in a dose-dependent manner. EGF at the concentration of 5 and 10 ng/ml did not seem to affect CO-029 protein expression. However, EGF at the concentrations of 25 to 100 ng/ml increased the expression level of CO-029 and its expression level reach a peak value at 100 ng/ml (P<0.01) (Fig. 3A).

The cells were also treated with 100 ng/ml EGF for 6 to 48 h (Fig. 3B) and the results demonstrated that EGF affected CO-029 protein expression in a time-dependent manner. EGF at a concentration of 100 ng/ml increased the expression level of CO-029 following 12 to 48 h of treatment (P<0.01). However, there was no significant difference observed between the 6-h treatment group and the control group (P>0.05).

The cells were treated with 100 ng/ml EGF for 12 to 72 h, and MTT assay was then performed to determine the effect of EGF on cell proliferation. We found that EGF promoted cell growth, and the cells treated with EGF proliferated at a higher rate compared with the untreated controls (Fig. 4).

To determine the effect of EGF on the invasion ability of the cells, the cells were treated with 100 ng/ml EGF for 24 h and Transwell-Matrigel invasion assay was then performed. Cell invasion assay revealed that the cells treated with EGF showed an enhanced invasion ability in comparison to the untreated controls (P<0.01) (Fig. 5).

The cells transfected with CO-029 siRNA (Si) or scramble control siRNA (Scr) were treated with 100 ng/ml EGF for 24 h followed by the determination of CO-029 protein expression levels by western blot analysis. CO-029 expression was successfully reduced by siRNA (P<0.01; Fig. 6).

MTT assay revealed that EGF promoted cell growth; however, the cells with a reduced expression of CO-029 proliferated at a lower rate compared with the cells transfected with the scramble control siRNA (Scr) in response to EGF treatment (Fig. 7).

We also examined whether CO-029 is involved in the effect of EGF on cell invasion. CO-029 siRNA was transfected into the cells for 24 h, and the cells were subsequently treated with 100 ng/ml EGF for a further 24 h. The results from the Transwell-Matrigel invasion assay revealed that the EGF-induced cell invasion was attenuated by the knockdown of CO-029 expression (P<0.05; Fig. 8).