Tissue samples were obtained from patients with a pathological diagnosis of NSCLC as determined by two pathologists. Patients were operated on in Department of Thoracic Surgery, the First Affiliated Hospital, Bengbu Medical College during the period from March 2005 to November 2007. There were 160 patients who signed informed consent forms to participate in the study, among them 120 NSCLC patients, 40 patients with benign pulmonary disease (36 chronic inflammation, and 1 case each of pulmonary haemorrhage, pulmonary fibrosis, inflammatory pseudotumor, and hamartoma). Patients received no chemotherapy or radiotherapy before operation. The patients characteristics: male 94, female 26; age 42–76, average 61 years old; histology: squamous cancer 100 cases, adenocarcinoma 20 cases; well-differentiated cancer 45 cases, moderately differentiated cancer 46 cases and poorly differentiated cancer 29 cases; TNM staging: 43 cases in stage I-II and 77 cases in stage III according to 1997 revised version of lung cancer staging standard by International Union Against Cancer (UICC). All of the patients had complete follow-up data and received conventional post-surgery chemotherapy. The principle committee of the First Affiliated Hospital of Bengbu Medical College had authorized this research.
Goat anti-human survivin monoclonal antibody and anti-human HIF-1α monoclonal antibody were purchased from Santa Cruz (Santa Cruz, CA, USA). Lipofectamine™ 2000 and Trizol were purchased from Invitrogen (Carlsbad, CA, USA). pGEM-T-EASY vector, pGL3-basic vector, pGL3-control vector, pRL-Tk vector and the Dual-Luciferase® Reporter Assay System were purchased from Promega (Madison, WI, USA). Universal Gene DNA Extraction Kit Ver.3.0, PrimeScript™ RT-PCR KIT, Agarose Gel DNA Purification kit2.0, Minibest Plasmid Purification kit 2.0, TaKaRa MutanBEST Kit, PrimerSTARTM HS DNA Polymerase, SYBR PrimeScript™ RT-PCR Kit were purchased from Takara BioTechnology Co., Ltd (Dalian, China). The dATP was purchased from Fermentas (Burlington, Canada). Primers were synthesized by Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China).
Cell line and culture
Human lung adenocarcinoma cell line A549 was maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (Invitrogen,), 100 units/ml of penicillin and 100 mg/ml of streptomycin (Invitrogen) in a humid atmosphere incubator with 5% CO2 at 37°C. To study the expression of survivin induced by hypoxia, A549 cells were incubated in hypoxic condition (1% O2, 5% CO2 and 94% N2) for 24 h.
Immunohistochemical staining using the streptavidin peroxidase method (S-P method) was performed on 4-μm sections of paraffin-embedded specimens to detect expression of survivin and HIF-1α protein in NSCLC and benign lung disease tissues. In brief, after deparaffinization and hydration, the slides were treated with endogenous peroxidase in 0.3% H2O2 for 30 min, after which the sections were blocked for 2 hrs at room temperature with 1.5% blocking serum in phosphate-buffered saline (PBS). Sections were then incubated with anti-Survivin antibody (1:200 dilution) or anti-HIF-1α antibody (1:200 dilution) at 4°C overnight., followed by washing in PBS, and incubation with secondary anti-mouse biotinylated antibody (1 : 2000) in PBS for 30 min at 37°C. Antibody binding was detected using the streptavidin-biotin-peroxidase complex/HRP, Code K0377 (Dako), with 3,3 diaminobenzidine for 3 min as a chromogenic substrate. Finally, the slides were lightly counterstained with hematoxylin. As a negative control, duplicate sections were immunostained without exposure to primary antibodies. The results were observed under a light microscope.
PCR-based Site Directed Mutagenesis of survivin promoter
Genomic DNA of A549 cells was extracted with Universal gene DNA extraction kit ver.3.0 according to the manufacturer's instructions. Survivin core promoter 230 bp (-203 ~ +27 bp) was amplified by PCR using primers with sequences selected from the survivin core promoter sequence; (Forward: 5'-ATC GAC GCG TTC TTT GAA AGC AGT CGA GGG GGC-3', Reverse: 5'-CCC AAG CTT TCT GGC GGT TAA TGG CGC GCC-3',). The cycling parameters were 95°C for 10s as a pre-denature step, followed by 40 cycles of 95°C for 5s, and 55°C for 30s, 72°C for 10 min. PCR products were purified, a polyadenylated by T4 DNA ligase, and then cloned to T-vector, named pGEM-T-EASY-sur230 bp. The template for site-directed mutagenesis was pGEM-T-EASY-sur230 bp. The forward and reverse primers (Forward: 5'-AGC GCT CCC GAC ATG CCC CGC GGC-3', Reverse: 5'-GCC CTCTTA GGC GGT CCA C-3') were used for PCR amplification. The cycling parameters were 30 cycles of 95°C for 10s, 60°C for 5s, 72°C for 30s. The linear product was self ligated after a blunting kination reaction; the product was named pGEM-T-EASY-sur229 bp and confirmed by sequencing.
Construction of survivin promoter-luciferase reporter vectors, and transfection into A549 cells
The mutant, and normal constructs were removed from pGL3-basic by restriction endonuclease Mlu I/Hind III. The reporter vectors were constructed by T4 DNA ligase, named pGL3-SVP-229-luc (mutant) and pGL3-SVP-230-luc (normal). A549 cells were plated onto 6-well plates one day prior to transfection. Following confirmation of 70%–80% confluence, the cells were transfected with pGL3-Basic without promoter (negative control), pGL3-SVP-229-luc (mutant plasmid), and pGL3-SVP-230-luc (normal plasmid). For cell transfection, A549 cells were transiently transfected with 2 μg plasmids and 0.2. g internal control plasmid pRL-TK by using Lipofectamine 2000™ reagent according to the manufacturer's instructions.
Luciferase reporter gene expression detection
Thirty hours after transfection, cells were harvested and lysed with 1 × lysis buffer (Promega), and then 20 μl of cell extract was assayed for luciferase activity using the Dual-Luciferase assay kit (Promega) according to the manufacture's instructions. The relative level of reporter gene expression was expressed as the ratio of firefly luciferase activity to Renilla luciferase (LU/RL).
A double strand siRNA oligonucleotide targeting HIF-1α (sense: 5-CUGAUGAC CAGCAACUUGAdTdT-3, antisense: 5-UCAAGUUGCUGGUCAU CAGdTdT-3) was designed based on the reference  and synthesized by Shanghai Genepharma Co. Ltd. (China). A pair of negative control siRNA were also designed with sequences different from siRNA-HIF-1α and not homologous to any sequences found in gene bank (sense: 5-AGUUCAACGACCAGUAGUCdTdT-3, antisense: 5-GACUACUGGUCGUUGA dTdT-3). For transfection, cells were plated onto 10 cm2 cell culture dishes and grown to 30–50% confluence before transfection. 50 μl of Oligofectamine transfection reagent per dish (Invitrogen) was added, and the cells were incubated at room temperature for 20 min. The cells were then rinsed with Opti-Mem I to remove any residual serum. The siRNA duplexes were diluted to a final concentration of 20 nM in Opti-Mem I (Invitrogen). Cells were incubated with the oligonucleotide duplexes in serum-free conditions for 4 h at 37°C. Serum was then added back to the culture, and cells were incubated in normoxic or hypoxic condition for an additional 48 h.
Real Time Reverse Transcription-PCR
Total RNAs were isolated using Trizol reagent (Invitrogen) according to the manufacturer's instruction. Twenty-five nanogram total RNA per sample was reverse transcribed by using the Reverse Transcription Reaction Kit (Takara Code: DRR061S) according to the manufacturer's instructions. Quantitative real-time PCR was performed analyzed on the Applied Biosystems 7300 Real-Time PCR System to determine the relative amounts of survivin, HIF-1α and GAPDH (internal control) mRNAs expressed. The SYBR Green Supermix was used for all real-time PCR reactions. The primers used in this study were: forward: 5'-AGCCA GACGATCAT GCAG CTACTA-3 ', and reverse: 5'-TGTGGTAAT CCACTTT CATC CAT TG-3 ' for HIF-1α PCR product (167 bp); forward: 5'-AGGTCATCTCGGCTGTTCCTG-3', and reverse: 5'-TCATCCTCACTGCGGCT GTC-3', for survivin PCR product (147 bp); and forward: 5'-GGTCTCCTCTGAC TTCAACA-3', and reverse: 5'-AGCCAAATTC GTTGTCATAC-3' for GAPDH PCR product (116 bp). The quantitative real-time PCR PCR parameters were 95°C for 10s as a pre-denature step, followed by 40 PCR cycles of 95°C for 5 s and 60°C for 30 s, and 72°C for 10 min. Data presented in this study was collected at 60°C applying a threshold of 0.002 and normalized to GAPDH using the default RQ ddCt study software.
Western Blot Analysis
After treatment, cells were washed two times with ice-cold PBS and then lysed by Cell Lysis Solution (DSL, USA). Cell lysates were incubated for 20 min at -20°C, and then centrifuged at 13,000 g for 20 min at 4°C. Supernatants were collected and protein concentration was determined by the Bradford method. Fifty microgram of protein from each sample was subjected to SDS-PAGE. After electrophoresis, proteins were transferred from the gel to polyvinylidene difluoride (PVDF) membranes (Millipore MA, USA). After blocking in a solution of 5% non-fat dry milk diluted in TBS, the membranes were washed, and incubated with primary antibodies [goat anti-survivin (1/200), rat anti HIF-1α (1/200), or rat anti-β-actin (1/800)] for 3 h at room temperature. After washing, the membranes were incubated with the appropriate horseradish peroxidase-labelled secondary antibody (1/2000) for 1 h. Blots were developed using a chemiluminescent detection system (ECL, Amersham Biosciences, Buckinghamshire, UK).
The samples were analyzed by Q test, analysis of variance and Chi-square tests to determine whether there were significant differences between individual groups. The correlation of survivin and HIF-lα protein in NSCLS was analyzed by Spearman correlation analysis. All the tests were performed using SPSS 11.5, and p < 0.05 was considered significant.