The Ishikawa cell line was kindly supplied by the Department of Pathophysiology, Beijing University. Cells were cultured in RIPM1640 with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin in an incubator maintained at 37°C and 5% CO2. Celecoxib, a selective COX-2 inhibitor, was purchased from Santa Cruz Biotechnology and dissolved in DMSO to generate a 100 mM stock solution that was stored at −20°C. For inhibition experiment, confluence cells were starved by serum deprivation overnight. Then, cells were treated with 80 μM celecoxib and incubated for 48 h.
Construction of pcDNA3.1-HER2/neu
Upstream (5′-TGGGAGCCTGGCATTTCTG-3′) and downstream (5′-TCCGGCC ATGCTGAGATGTA-3′) primers were designed based on HER-2/neu cDNA sequence obtained from GenBank. For cloning, HindIII/XbaI restriction endonuclease sites were inserted flanking the target gene primers. Primers were synthesized by TaKaRa Biotechnology Co., Ltd. Total RNA was isolated from Ishikawa cells using TRIzol reagent (TaKaRa, China) according to the manufacturer’s instructions. HER-2/neu cDNA was reverse-transcribed using the One Step RNA PCR Kit (TaKaRa) according to the manufacturer’s recommendations. PCR conditions included denaturation at 94°C for 5 min, 25 cycles of denaturation at 94°C for 45 s, annealing at 60°C for 1 min, and extension at 72°C for 6 min, with a final extension at 72°C for 10 min. PCR products were separated on 1% agarose gel and eluted. The PCR product was sent to TaKaRa for sequencing. PcDNA3.1 plasmid and HER2 cDNA were digested with HindIII/XbaI double endonucleases. The digested products were separated by agarose gel electrophoresis and purified. Pure HER2 cDNA and vector were mixed at a 4:1 ratio and were ligated at 16°C for 20 h. Ligation products were transformed into E. coli. After culturing at 37°C overnight, E. coli cells were screened. DNA was isolated from positive colonies (named pcDNA3.1 (+)-HER2), and were sequence-verified.
Gene transfection and G418 screening
pcDNA3.1 (+)-HER2 was isolated using a plasmid extraction kit (Invitrogen, USA) according to the manufacturer’s protocol. Ishikawa cells in logarithmic growth were transferred to 6-well culture plates at 1 × 106 cells/well and were cultured for 1 d prior to transfection. Ishikawa cells then were transfected with pcDNA3.1-HER-2/neu or pcDNA3.1 (control) using Lipofectamine2000 (Invitrogen, USA) according to the manufacturer’s protocol. Non-transfected cells were cultured as the negative control. The original culture media was discarded 3 d after transfection, and cells were cultured in complete media containing 10% fetal bovine serum and 700 μg/ml of G418 (Invitrogen, USA). G418-resistant clones were selected and transferred to culture media containing 350 μg/ml of G418 for scale-up culture.
To knock down the HER2/neu in Ishikawa cells, the siRNA transient transfection experiment was performed according to the previous publication . Briefly, Ishikawa cells were transfected with 25 nM COX-2 siRNAs, respectively. Non-targeting siRNA was used as negative control. Transfections were carried out according to the guidelines for the DharmaFECT® siRNA Transfection Reagents (Dharmacon). Ishikawa cells were collected at 72 hours post-siRNA addition for protein western blotting analysis.
Real-time RT-PCR analysis of HER-2/neu
Total RNA was extracted from Ishikawa cells stably transfected with pcDNA3.1 (+)-HER2 using TRIzol, as above. The same HER-2/neu primers were used as in the construction of pcDNA3.1 (+)-HER2. GAPDH was used as the internal control (upstream 5′-CATCCATGACAACTTTGGTATC-3′; downstream 5′-CCATCACGCCACAGTTTC-3′). cDNA was synthesized from 1 μg of total RNA using oligo(dT) primers in the presence of reverse transcriptase. Gene amplification was performed on a real-time PCR instrument (TaKaRa, China) using 1 μl of cDNA as template in a 25 μl volume. PCR was started at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 10 s, annealing at 59°C for 15 s, and extension at 72°C for 20 s, with a final extension at 72°C for 10 min. Fluorescence intensity was monitored and recorded in real time. A melting curve analysis was performed after amplification was complete. The ΔΔCt value was used to evaluate expression levels of HER2 and COX-2 mRNA. By this method, a higher expression level is related to a lower ΔΔCt value.
Western blotting for HER-2/neu, COX-2, and P450arom
Cells collected from non-transfected, pcDNA3.1-transfected, and pcDNA3.1-HER2-transfected groups were lysed with 250 μl protein extracting fluid (RIPA lysis buffer: 50 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS), homogenized for 10 min, incubated in an ice-bath for 1 h, and centrifuged at 12,000 × g for 30 min at 4°C. Supernatants were collected, and protein concentrations were determined using the BCA protein assay system (Pierce, USA). Proteins were separated by 12% SDS-PAGE and were transferred to PVDF membranes. After blocking overnight at 4°C in 1 × PBS, 0.1% Tween 20, and 5% non-fat milk, membranes were incubated with anti-HER-2/neu (1:800), COX-2 (1:400), P450arom (1:400) and β-actin (1:800) polyclonal antibodies (Santa Cruz Biotechnology, USA) for 3 h at room temperature. Membranes were washed twice and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ZhongShan, China, 1:1,500) for 2 h at room temperature. Immunodetection was performed by chemiluminescence (ECL reagent, Beyotime, China) and membranes were exposed to film. Images were captured using a transmission scanner. For quantification, target proteins were normalized to β-actin (the internal standard) by comparing the gray-scale values of proteins to corresponding β-actin values. Quantification was performed using UVP Gelworks ID Advanced v2.5 software (Bio-Rad, USA).
ELISA for PGE2 and E2 detection
Supernatants were collected from non-transfected, pcDNA3.1-transfected, and pcDNA3.1-HER2-transfected groups for ELISA. Supernatant PGE2 and E2 concentrations were measured using an ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Each sample was examined in triple and averaged for data analysis.
SPSS v10.0 software was used for all statistical analyses. Data were expressed as mean ± standard error of the mean (SEM). One-factor analysis of variance was used for pairwise comparison. Statistical significance was defined as P < 0.05.