Cell culture and hypoxia treatment
The human gastric cancer cell line SGC-7901 was obtained from the China Center for Type Culture and was maintained in RPMI-1640 medium (Hyclone, Beijing, China) containing 10% fetal bovine serum (Sijiqing Bio. 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 at 70–80% of confluency for experimental purposes. After seeding for 24 h, cells were incubated in normal or hypoxic conditions for a further 48 h (for hypoxic treatment, cells were maintained in a hypoxia incubator of 1% oxygen concentration infused with 5% CO2 and nitrogen gas mixture).
RNA interference and groups
Two pairs of siRNAs were designed against human HIF-1α and survivin, as follows: siRNA-HIF-1α, 5'- CCAUAUAGAGAUACUCAAATT-3' (sense) and 5'-UUUGAGUAUC UCUAUAU GGTT-3' (antisense); siRNA-survivin, 5'-GGCUGGCUUCAUCCACUGCTT-3' (sense) and 5'- GCAGUGGAUGAAGCCAGCCTT-3' (antisense).
The sequences 5'-UUGAUGUGUUUAGUCGCUATT-3' (sense) and 5'-UAGCGACUAAACACAUCAATT-3' (antisense), named scrambled siRNA (SCR) were used as a negative control. 3′-Fluorescein amidite (FAM) fluorescence-labeled SCR was utilized to detect the transfection efficiency. All siRNAs were chemically synthesized by Gen. Co., Ltd. (Shanghai, China).
The cultured cells were randomly divided into four groups: a blank control group, a siRNA-Survivin group (ss group), a siRNA-Survivin+siRNA-HIF-1α group (sis+siH group) and a SCR control group. For siRNA transfection, SGC-7901 cells were prepared on 6-well plates and incubated overnight to achieve 80-90% confluency. The cells were transfected with 100 nmol/L siRNAs using HifectinⅡ (Applied Gen Co., Ltd., Beijing, China) according to the protocol provided by the manufacturer. Transfection efficiency rates＝cells displaying green fluorescence／total count cells×100％. Subsequently, the exponentially growing SGC-7901 cells were maintained under hypoxic conditions for further experiments. Three independent repeats were conducted for all experiments.
Cell viability assay
The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) (Funakoshi Co., Tokyo, Japan) assay was performed to evaluate cell proliferation. SGC-7901 cells were seeded on 96-well plates at an optimal density (3×103 cells per well) and incubated overnight. After another 48-h incubation in normal or hypoxic conditions, cells were treated with 20 µL MTT (5 mg/mL) and 150 µL dimethyl sulfoxide (DMSO). The optical density (OD) value of each well was measured at 490 nm using a 2100C ELISA Reader (Rayto Sciences Co., Ltd. Shanghai, China). The cell viability curve of the four groups was drawn. The cell proliferation inhibition rate = (1-average OD value experimental group/average OD value control group) × 100%.
Reverse transcription polymerase chain reaction (RT-PCR)
The primers were as follows: sense, 5'-GCAAGCCCTGAAAGCG-3' and antisense, 5'-GGCTGTCCGACTTTGA-3' for HIF-1α (240 bp); Sense, 5'-AACAGCCGAG ATGACCTCC-3' and antisense, 5'-AACTTCAGGTGGATGAGGAGAC-3' for survivin (421 bp); GAPDH (glyceraldehyde-3-phosphate dehydrogenase) sense, 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and antisense, 5'-CATGTGGG -CCATGAGGTCCACCAC-3' for the internal reference (983 bp). Total mRNA was isolated with TRIzol® reagent (Invitrogen Life Techonology, USA), and cDNA was prepared with the GoScript™ Reverse Transcription System kit (Promega Biotech Co., Ltd., USA) according to the manufacturer's instructions. The PCR program was as follows: denaturation at 95˚C for 5 min; 30 cycles of 94˚C for 30 s, annealing at 50˚C for 30 s and 72˚C for 1 min, with a final step at 72˚C for 5 min. PCR products were separated using 2% agarose gel, and the bands were scanned and the relative mRNA expression levels were determined by comparing with the expression of GAPDH.
Western blotting analysis
After treatments, total protein was extracted from cells using radio -immunoprecipitation assay (RIPA) buffer (Beyotime Biotech Co., Ltd., Wuhan, China) and the level of protein was determined using the bicinchoninic acid assay method. Equal amounts of protein lysates (50 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (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 and then incubated at 4˚C overnight with monoclonal rabbit anti-mouse antibodies against HIF-1α, GAPDH (Boster Co., Ltd., Wuhan, China) and survivin (Biosynthesis Co., Ltd., Beijing, China). On the following day, the membranes were washed with TBST and incubated for 1 h at room temperature with the secondary peroxidase-labeled goat anti-rabbit antibody (Boster Co., Ltd, Wuhan, China) diluted to 1:2000 in skimmed milk/TBST. The protein bands were visualized by enhanced chemi-luminescence and the band intensity was measured using Quantity One v4.6.2 software (Bio-Rad, California, USA). Quantitative analysis of the relative levels of target proteins was determined using the NIH ImageJ software.
Flow cytometry
For analysis of apoptosis, an annexin V-FITC apoptosis detection kit was used (Invitrogen, USA). After treatment for 48 h, SGC-7901 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 min at room temperature away from light. The apoptotic rates were calculated based on the number of these transfected gastric cancer cells in the apoptotic state and evaluated immediately by flow cytometry (BD Biosciences, Franklin Lakes, NJ) . The mean fluorescence intensity of annexin-V-FITC/PI was determined by flow cytometry. Then the apoptotic rates were calculated at the mean fluorescence intensity. At last, the results were quantified using WinMDI 2.9 analysis software.
Wound healing assay
Scratch (wound‑healing) assays were performed to determine cell migration ability. SGC-7901 cells were seeded in plates at a density of 3×105 cells/well and incubated overnight before being treated as described above. After 24 hours, cells were maintained in normoxic or hypoxic conditions and scratched using a sterile 200-μL tip to create a wound. Cells were then washed three times with PBS to remove debris, and were cultured in a serum-free medium for further 24 hours. The width of the wound area was monitored and measured at more than three positions per scratch by using microscopy to compare the migration ratios among the groups.
Invasion assay
Matrigel invasion assays were employed to assess the invasion of SGC-7901 as previously described. Treated cells were incubated overnight in the serum-free medium. Then, 50 μL Matrigel (BD Biosciences, San Jose, CA) was overlaid and maintained at 37 °C for 1 hour inside transwell filters with a membrane pore size of 8.0 μm. Next, 5×104 of treated cells suspended in the serum-free RPMI-1640 medium were added in the upper chambers and the bottom chamber of each well contained only RPMI-1640 medium with 10 % FBS. After incubation in normoxic or hypoxic conditions for 12 h, cells on the upper surface were removed using a cotton swab. 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 the microscope, and the relative numbers were counted (five distinct fields per insert).
Statistical analysis
Data were expressed as mean values with standard error of the mean (± SEM). Statistical analysis was performed using Student’s t test with SPSS11.0 for Windows. All experiments were performed in triplicate. Differences were considered statistically significant at P < 0.05.