Cell culture and cell lines
The cell lines GES-1, MKN-28, MKN-45, BGC-823, NCL-N87, SNU-16, SGC-7901 and AGS were cultured in RPMI-1640, supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA). They were cultured in an atmosphere of 5% CO2 at 37°C. BGC-823, NCL-N87 and AGS cells were obtained from Shanghai Institute of Cell Bank (Shanghai, China); GES-1, MKN-28, MKN-45, SNU-16 and SGC-7901 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).
Amplification of the ERas full-length transcripts and sequencing analysis
Total cellular RNA was extracted from each cell line with TRIzol® (Invitrogen Life Technologies, Carlsbad, CA, USA), and cDNA was synthesized with Reverse Transcription kit (Promega Corp., Madison, WI, USA), and both were performed according to the manufacturer’s protocol. The cDNA was synthesized by PCR with PrimeSTAR HS DNA polymerase (Takara Bio, Shiga, Japan). The primers used for the full-length ERas coding sequence were: forward, 5′-atggagctgccaacaaagcctggca-3′ and reverse, 5′-ttcaggccacagagcagccacagt-3′, which gave an amplified fragment of 702 bp. The reaction conditions were: 94°C for 15 sec, 62°C for 45 sec and 72°C for 30 sec, repeated for 35 cycles. The products were separated by 1.2% agarose gel electrophoresis, and the 702 bp bands were recycled using aqua-Spin gel extraction mini kit (Watson Biotechnologies, Inc., Shanghai, China). The identification of PCR products was confirmed by sequencing analysis (Songon Biotech Co., Shanghai, China).
Real-time quantitative PCR
The real-time quantitative PCR analyses were performed in triplicate using SYBR® Premix Ex Taq™II kit (Takara Bio). The GAPDH gene was chosen as an endogenous control. The primers used for ERas were: forward, 5′-cacatggagcccttccttc-3′ and reverse, 5′-tgtccagggtcaactccttc-3′; the primers used for GAPDH were: forward, 5′-ggacctgacctgccgtctag-3′ and reverse, 5′-gtagcccaggatgcccttga-3′; the PCR conditions were as follows: 94°C for 15 sec, 58°C for 45 sec, 72°C for 20 sec, repeated for 35 cycles. Amplified products were separated by 0.9% agarose gel electrophoresis.
Western blot analysis
Cells were lysed in a lysis buffer containing 2% sodium dodecyl sulfate (SDS) and 0.125 M Tris-HCl (pH 6.8) on ice for 30 min, followed by high-speed centrifugation, and the supernatant protein was finally collected. SDS-PAGE was performed using 10% polyacrylamide gels. PAGE separated proteins were electrophoretically transferred onto nitrocellulose membranes. The membrane filters were blocked with 5% powdered milk in TBST (0.1% Tween-20) for 2 h and then incubated in rabbit ERas antibody (Abgent, Suzhou, China) diluted 1:100 in TBST at 4°C overnight, and finally incubated with HRP anti-rabbit secondary antibody (Kangchen Biotech, Shanghai, China) diluted 1:2,000 for 1 h at room temperature. Antigens on the membrane were detected with enhanced chemiluminescense detection reagents (Roche, Basel, Switzerland).
Small interfering RNA transfection
Two ERas stealth siRNA, no. 30 forward, GCAACUAGCUUUGAGGGAC(dTdT) and reverse, GUCCCUCAAAGCUAGUUGC(dTdT); no. 32 forward, GUAACAUGGGAGUGCCUAA(dTdT) and reverse, UUAGGCACUCCCAUGUUAC(dTdT); one high GC% negative control siRNA forward, CCUACGCCACCAAUUUC GU(dTdT) and reverse, ACGAAAUUGGUGGCGUAGG (dTdT) were designed and synthesized (Bioneer, Daejon, Korea). siRNA was mixed with Lipofectamine™ 2000 (Invitrogen Life Technologies) in an OptiMEM serum-free medium (HyClone) for 30 min at room temperature and then added to each 24-well plate containing MKN-28 or BGC-823 cells. Cells were maintained in a humidified 5% CO2 incubator at 37°C for 6 h with the old medium being replaced by a fresh medium. After 24 h of transfection, cells were harvested for cell proliferation, migration and colony formation assays.
siRNA-transfected MKN-28 and BGC-823 cells were seeded into 96-well plates at a density of 3×103 cells/well and maintained in culture medium for 5 days. Each well set five duplicates. We measured cell growth using cholecystokinin (CCK) assay by Cell Counting Kit (Dojindo, Tokyo, Japan) according to the manufacturer’s instructions.
Cell migration assay
For wound-healing experiments, MKN-28 and BGC-823 cells transfected with siRNA were cultured to 80% confluence after being seeded into 6-well plates, then scraped using a p10 tip (time 0), and suspended cells were washed with PBS three times. Cells were incubated for another 4 days and images were captured by microscope (Zeiss, Oberkochen, German) at the same time every day. Migration distance was measured from images (5 fields) at each indicated time point.
Transwell assay of MKN-28 and BGC-823 cells was assessed using 6.5 mm diameter inserts (Corning Costar Corp., Corning, NY, USA). A total of 3×104 cells were suspended in 100 μl serum-free RPMI-1640 medium and loaded into upper wells; lower chambers were filled with 600 μl of complete medium (RPMI-1640 supplemented with 10% FBS). Migration chambers were incubated in a humidified 5% CO2 incubator at 37°C for 24 and 48 h. Cells were then fixed with 600 μl of paraformaldehyde for 20 min. The inner surfaces of the upper chambers were wiped using cotton swabs to remove non-migrated cells in the migration assay. The chambers were then washed with PBS and stained with 500 μl crystal violet for 20 min at room temperature. Stained cells were counted using the ImageJ software, and 5 random fields were counted (Zeiss).
Colony formation assay
A total of 500 siRNA-transfected MKN-28 and BGC-823 cells were seeded in 6-well plates and incubated for 14 days respectively, with the medium replaced every 4 days. On the 15th day, the cells were stained with crystal violet for 20 min and washed with tap water for 10 min. For each dish, colonies in five random fields were counted using the ImageJ software.
Each measurement was performed in triplicate. Original real-time PCR data, CCK-8 data, migration/invasion data and colony formation data were recorded as continuous variables and analyzed using Student’s t-test or linear polynomial ANOVA with LSD post hoc examination. All statistical analyses were performed using SPSS 16.0 software. P-values <0.05 were considered to indicate statistically significant differences.