1.1 Cell culture and hypoxia treatment
The Human gastric cell line BGC-823 were obtained from the China Center for Type Culture and maintained in RPMI 1640 media (Hyclone, Beijing, China) containing 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China) and 1% penicillin/streptomycin, in a 5% CO2 humidified atmosphere at 37°C incubation. The medium was changed at alternate days and cells were harvested in 70–80% of confluence for exprement. 24 hours after seeding, the cells were incubated in normal or hypoxia condition for another 48 h (For hypoxia treatment, cells were maintained in a hypoxia incubator of 1% oxygen concentration infused with 5% CO2 and nitrogen gas mixture).
1.2 RNA interference
Two pairs of siRNAs designed against human HIF-1α and survivin separately, valid and described by others previously, were employed in the present experiment. The nucleotide names and sequences were as follows: siRNA-HIF-1α, 5'- CCAUAUAGAGAUACUCAAATT-3'(sense), and 5'-UUUGAGUAUCUCUAUAU GGTT-3'(antisense); siRNA-survivin, 5'-GGCUGGCUUCAUCCACUGCTT-3' (sense), and 5'- GCAGUGGAUGAAGCCAGCCTT-3' (anti sense). To avoid triggering innate immune responses of the siRNA, the sense strands of were 2'-O-methyl (2'OMe) uridine modified, while the anti-sense strands were not modified. Meanwhile, the sequences 5'-UUGAUGUGUUUAGUCGCUATT-3' (sense), and 5'-UAGCGACUAAACACAUCAATT-3' (antisense), named SCR (scrambled siRNA), which has no homology to human genes, was used as a negative control. 3'‑fluorescein amidite (FAM) fluorescence‑labeled SCR was used to detect the transfection efficiency. All siRNAs were chemically synthesized by Shanghai Genepharma Co. Ltd.(China). For siRNA transfection, BGC-823 Cells were seeded on 6-well plates at a density of 1×105 cells/well and incubated overnight to reach 80% confluence, then the cells were transfected with 100 nM siRNAs using HifectinⅡ (Beijing Applygen Co.Ltd., Beijing, China) according to the protocol provided by the manufacturer. Subsequently, the exponential growth BGC-823 cells were maintained in hypoxia conditions for the further experiments.
1.3 Cell Viability assay
The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT, Roche Diagnostics Corporation) assay was performed to evaluate the cell proliferation. In brief, BGC-823 cells were seeded onto 96-well plates at an optimal density (3×103 cells per well) and incubated overnight, then the treatment mentioned above were performed respectively. After another 48h incubation in normal or hypoxia condition, the cells were treated with 20 µL MTT (5 mg/ml). The cultures were then re-incubated for an additional 4 h. After removal of the supernatant, 150 µL DMSO was added to each well to dissolve the crystals completely and then the absorbance was measured at 490 nm using a 2100C ELISA Reader (Rayto Life and Analytical Sciences Co., Ltd. Shenzhen, China).
1.4 Reverse transcription-PCR (RT-PCR) analysis
The sequences of primers were synthesized by TAKARA Biotechnology (DaLian, China) Co., Ltd.: Sense, 5'-GCAAGCCCTGAAAGCG-3' and antisense, 5'- GGCTGTCCGACTTTGA-3' for HIF-1α (240‑bpproduct);Sense,5'-AACAGCCGAGATGACCTCC-3' and antisense, 5'- AACTTC AGGTGGATGAGGAGAC-3' for survivin (421‑bpproduct);and sense, 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and antisense, 5'-CATGTGGGCCATGAGGTCCACCAC-3' for the internal reference, GADPH (983-bp product). After total mRNA were isolated with the TRIzol® reagent (Invitrogen, USA) and cDNA was prepared with the GoScript™ Reverse Transcription System kit (Promega Biotech Co., Ltd., Beijing, China) according to the manufacturer's instruction, RT-PCR was performed. The PCR reaction condition was as follows: 95˚C denaturation for 5 min; 30 cycles of 94˚C for 30 sec, 50˚C annealing for 30 sec and 72˚C for 1 min, with a final step at 72˚C for 5 min. PCR products were separated in 2% agarose gel. The bands were scanned and relative mRNA expression levels were determined by comparing with the expression of GAPDH.
1.5 Western bloting analysis
Total protein was extracted from cells using RIPA buffer (Beyotime Institute of Biotechnology) after treatment and the level of protein was determined using the bicinchoninic acid assay method. Equal amounts of protein lysates (50 μg) were separated by SDS–PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore Co., Hayward, CA, USA), the membranes were blocked with 5% skimmed milk for 1 h. Then the membranes were incubated at 4˚C overnight with monoclonal rabbit anti-mouse antibodies against HIF-1α, GADPH (Boster Biological Engineering Co., Ltd., Wuhan, China) and survivin (Biosynthesis Biotechnology co., Ltd., Beijing. China) , respectively. The next day, the membranes were washed with TBST and incubated with the secondary peroxidase-labeled goat anti-rabbit antibody (Boster Biological Engineering Co., Ltd) diluted to 1:2000 in skimmed milk/TBST for 1 h at room temperature. The protein bands were visualized by enhanced chemiluminescence and the band intensity was measured using Quantity One v4.6.2 software (Bio-Rad Laboratories, California, USA).
1.6 Flow cytometry
For cell apoptosis rate analysis, the annexin V-FITC apoptosis detection kit was used (Invitrogen). 48 h after treatment, BGC-823 cells were harvested, washed twice with PBS, and resuspended in 500 μL of binding buffer, Cell suspensions were then incubated with 5 μL of annexin V-FITC and 5 μL of propidium iodide (PI) for 10 minutes at room temperature away from light. The cells were evaluated immediately by flow cytometry (BD Biosciences, Franklin Lakes, NJ). The results were quantitated using winMDI 2.9 analysis software.
1.7 Wound-Healing Assays
Scratch wound‑healing assays were performed to determine the cell migration ability. Briefly, lines were marked on the backs of six‑well plates, BGC-823 cells were seeded in it at a density of 3×105 cells /well and incubated overnight. Then treatments mentioned above were performed separately. 24 hours later, maintained in normal or hypoxia condition, treated cells were scratched by a sterile 200-μL tip perpendicular to the lines to make wound, washed three times with PBS to remove cell debris, and cultured in serum-free medium for another 24 hours. The width values of the wounded area was monitored and measured at more than three positions per scratch by microscopy to compare the migration ratios among the groups.
1.8 Invasion assays
Matrigel invasion assays were employed to assess BGC-823 cell invasion ability as previously described. In brief, treated cells were incubated overnight in serum-free medium. 50 μl Matrigel (BD Biosciences, San Jose, CA) was overlayed and maintained to solidify at 37°C for 1 hour inside transwell filters with membrane pore size of 8.0 μm (Corning Inc). 5×104 of treated cells suspended in serum free RPMI-1640 medium were added in the upper chambers, whereas the bottom of each well contained RPMI-1640 medium with 10 % FBS. After incubation in a normal or hypoxia condition for 12 hours, the cells on the upper surface were removed using a cotton swab, and the cells on the lower surface of the membrane were fixed (4 % paraformaldehyde) and stained with 0.1 % Crystal Violet. Cells on the lower surface of the filter were visualized and photographed under microscope and the relative numbers were counted (five distinct fields per insert).
1.9 Statistical analysis
The data were expressed as mean±standard error of the mean (SEM). Statistical analysis was analyzed by t test, using the statistical program, SPSS11.0 for Windows. All experiments in vitro were performed in triplicate. P<0.05 or less was considered statistically significant.