The NCI-H446 cell line was obtained from the American Type Culture Collection (CAS Shanghai Institutes for Biological Sciences cell bank) and cultured in RPMI-1640 medium (Sigma-Aldrich Co, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 100 μg/ml kanamycin at 37°C in humidified atmosphere containing 5% CO2 and 20% O2. The medium was routinely changed 2-3 d after seeding. All experiments were performed using exponentially growing cells and repeated at least 3 times.
Adenoviral construction and cell transfection
We used Ad5 (full name: tumor-specific replication-defective adenovirus type 5) as the vector. Ad5- HIF-1alpha, Ad5-siHIF-1alpha, Ad5-SOCS1 and Ad5-siSOCS1 were constructed and gifted from the Viral-Gene Therapy department of Shanghai Eastern Hepatobiliary Surgery Hospital. The cells in the microarray analysis were divided into five groups: control group (cells cultured in a normoxic environment with 20% O2), hypoxia group (cells cultured under a hypoxic environment with 1% O2), Ad5 group (cells transfected with Ad5), Ad5-HIF-1alpha group (cells transfected with Ad5-HIF-1alpha) and Ad5-siHIF-1alpha group (cells transfected with Ad5-siHIF-1alpha and cultured under hypoxic environment with 1% O2). For transfection, cells were cultured in 6-well plates and exposed to viral supernatant in the absence of cytokines and serum with different multiplicities of infection (MOIs): the number of plaque-forming units (pfu) per cell. The efficiency of transfection was estimated by determining the percentage of enhanced green fluorescence protein (EGFP)-positive cells in cells infected with Ad5-EGFP. To establish optimal conditions for NCI-H446 cells by adenoviral gene transfer, different titers of Ad5-EGFP were used. After transfection for 3 days, half of the virus-containing medium was replaced for the first time, and then plates were further incubated and all the medium was changed every 2 days. According to a report by Meng Jiang , we imitated the hypoxic microenvironment in vivo by putting the cells into a hypoxic chamber with an auto purge airlock (Thermo Forma, Tri-tube, USA). Environmental hypoxic conditions were established in an airtight humidified chamber that was continuously flushed with a gas mixture containing 1% O2, 5% CO2 and 94% N2 at 37°C.
RNA extraction, Microarray hybridization and data analysis
All the cells were washed gently with ice-cold phosphate-buffered saline (PBS) and lysed with 3 ml Trizol (Invitrogen, San Diego, CA, USA). According to the manufacturer's protocol, total RNA was extracted and purified with the RNAeasy kit (Qiagen, USA). The concentration of total RNA was measured by a Biophotometer (Eppendorf, Germany), and the quality of purified RNA was confirmed by agarose gel electrophoresis using ethidium bromide staining. cDNA was synthesized from each RNA sample using SuperScript System (Invitrogen) as a template for the preparation of biotin-labeled cRNA according to the GeneChip IVT Labeling Kit. The hybridization fluid was prepared and Biotin-labeled cRNA was hybridized to the GeneChip Human Genome U133 Plus 2.0, washed, stained with phycoerythrin-streptavidin and scanned according to the manufacturer's protocol. The microarray contained 54,614 human gene probe sets, each of which consisted of 11 probe pairs corresponding to a single mRNA transcript. After being saved as raw image files, all the data were converted into probe sets and analyzed by the GCOS software based on the method of normalization. Annotation by Unigene database http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db_unigene, gene number, gene symbol, and gene description were carried out using the database http://david.abcc.ncifcrf.gov/summary.jsp and Affymetrix databases. The results are presented as the ratios of the hypoxia group vs. control (normoxia) group, Ad5-HIF-1alpha group vs. Ad5 group1 and Ad5-si HIF-1alpha group vs. Ad5 group2. Ratio values with an increase or decrease of more than 2 folds were defined as differential expression. The primary data sets are all available at http://www.hopkins-genomics.org/expression.html.
Selecting genes for real-time quantitative PCR
The microarray data were verified by real-time quantitative PCR. Six upregulated genes were selected to validate and PCR primer pairs were as follows:
human IGFBP5: sense 5'-TGCCCAGAAAATGAAAAAGG-3' and
antisense 5'-GGATGACACAGCGTGAGAGA -3'
human IRS4: sense 5'-TACGGCAATGGCTTTATCAC-3' and
human TNFAIP6: sense 5'-TTTCAAGGGTGCCAGTTTCG-3' and
human SOCS1: sense 5'-TAGCACACAACCAGGTGGCA-3'and
human IL-6: sense 5'-CGGGAACGAAAGAGAAGCTCTA-3' and
antisense 5'- CGCTTGTGGAGAAGGAGTTCA-3'
human VEGF-A: sense 5'- CCATGAACTTTCTGCTGTCTT-3' and
Five downregulated genes were selected to validate and PCR primer pairs were as follows:
Human IGFBP3: sense 5'-GACGTATCTAGCAGCTGTCT-3'and
antisense 5'- CGAGGTCTCATGATCTCTCT -3'
Human ZNF569: sense 5'-GGAAAGAAACGACTGGGAGC-3' and
Human SOCS-2: sense 5'-CCTTTATCTGACCAAACCGCTCTA-3'and
Human SIRPa: sense 5'-GGCGGGTGAGGAGGAGCTGCAGGTGAT-3' and
Human XRCC4: sense 5'-AAGATGTCTCATTCAGACTTG-3'and
Real-time PCR was performed using SYBR ExScript RT-PCR Kit according to the manufacturer's protocol (Takara Biotechnology (Dalian) Co. Ltd., Dalian, China) and using the iCycler Real-Time PCR Detection System (BioRad). All the RNA samples, which were chosen from the microarray samples, were run in duplicate on 96-well optical PCR plates. The thermal cycling conditions were as follows: 1 cycle of 95.0°C for 10 min; 40 cycles of 95.0°C for 5 s; 60.0°C for 30 s; and 81 cycles of 55.0°C for 10 min (with an increase set point temperature after cycle 2 by 0.5°C). GAPDH was used as an internal control. The primers used for SYBR Green real-time PCR were designed according to the NCBI website http://www.ncbi.nlm.nih.gov and were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. The relative changes in gene expression were calculated using the equation: relative changes in gene expression = 2-ΔΔCTwhere ΔCt = Cttarget- CtGAPDH and ΔΔCt = ΔCtAd5-HIF-1alpha-ΔCtAd5, ΔΔCt = ΔCtAd5-ΔCtAd5-siHIF-1a or ΔΔCt = ΔCtHypoxia-ΔCtnormoxia
Western blot analysis
The cells of each group were washed with mild PBS to remove the remaining medium, and cellular proteins were extracted by disrupting cells in RIPA lysis buffer (Beyotime Institute of Biotechnology): 50 mM Tris pH 7.4, 1 mM EDTA, 250 mM NaCl, 0.1% NP40, 1% Triton X100, 0.5% SDS, 0.25% DOC, 1 mM NaF, 5 mM NaVO3, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, and 1 mM PMSF. The protein was electrophoresed on 12% SDS-polyacrylamide gels and transferred to a PVDF membrane. The membranes were then blocked at room temperature for 1 h with 5% non-fat milk in Tris buffered saline containing Tween20 (TBST). The rabbit anti-human primary antibodies (Wuhan Boster Biological Engineering Technology Limited Company) that detect IGFBP5, SOCS1, IL-6 and STAT3(Signal Transducer and Activator of Transcription 3)were incubated with membranes overnight at 4°C. The membranes were subsequently incubated with goat anti-rabbit peroxidase-conjugated secondary antibodies, and immunoreactivity was detected by using an enhanced Chemiluminescence kit and captured on X-ray film. β-actin was used as an internal control.
Analysis of the effect on cell growth and apoptosis by HIF-1alpha and SOCS1
In this study, all cells were divided into 7 groups: Ad5 group - transfection with Ad5 (control group); Ad5-HIF-1a group - transfection with Ad5-HIF-1 alpha; Ad5-si HIF-1alpha group - transfection with Ad5-siHIF-1alpha; Ad5-SOCS1 group - transfection with Ad5-SOCS1; Ad5-siSOCS1 group - transfection with Ad5-siSOCS1; Ad5-HIF-1alpha/siSOCS1 group - co-transfection with Ad5-HIF-1alpha and Ad5- siSOCS1; Ad5-siHIF-1alpha/SOCS1 group - co-transfection with Ad5-siHIF-1 alpha and Ad5-SOCS1; Ad5-HIF-1alpha/SOCS1 group - co-transfection with Ad5-HIF-1 alpha and Ad5-SOCS1. NCI-H446 cells of each group were prepared as a cell suspension and plated at a density of 1 × 104 cells/well into 6-well plates. Every 24 h, 3 wells were trypsinized for cell counting and repeatedly counted for 7 d to draw the growth curve. Then, cells of each group were washed with PBS and fixed in 70% ethanol for 24 h at 4°C. The fixed cells were resuspended in PBS. After incubation for 10 min, the apoptotic rates were analyzed by terminal transferase dUTP nick-end labeling (tunel stain)and all the procedures were performed according to tunel kit's protocol(Beyotime Institute of Biotechnology). After DAB coloration we began to calculate the apoptosis rate by using the formula: apoptosis rate = number of tunel positive cells/number of total cells.
All experiments were carried out in triplicate. Student's t test or ANOVA was used to compare parameters between the different study groups. A P value of less than 0.05 was considered statistically significant. The statistical analyses were performed with the Windows SPSS 13.0 package.