Patients and tissue samples
For this retrospective study, archival formalin-fixed, paraffin-embedded specimens from 230 primary NPC patients admitted from 1992-2002 to the Sun Yat-Sen University Cancer Center (Guangzhou, China) were recruited. All NPC samples were obtained before treatment with standard curative radiotherapy, with or without chemotherapy. Sixty patients were diagnosed as differentiated non-keratinized (WHO types II), and 170 patients had undifferentiated carcinoma (WHO type III). According to the Chinese 1992 staging system , patients were classified as 6 in stage I, 49 in stage II, 110 in stage III, 65 in stage IV. The majority of patients were male (173 of 230, or 75.2%), ranging from 86 to 14, with a median age of 46. This study was approved by the Research Ethics Committee of the Sun Yat-Sen University Cancer Center (Reference number: YP2009167).
Tissue microarray construction
Paraffin-embedded specimens were from a previously constructed tissue microarray. Protocols and instruments for the tissue array construction were described previously .
The immunohistochemistry (IHC) protocol was as described previously . Briefly, tissue sections were de-waxed for antigen retrieval, and incubated with primary antibodies LMP1 monoclonal antibody (CS 1-4, Neomarkers, USA) at a dilution of 1:50, or p-mTOR (Ser2448), p-P70S6K (Thr389), or p-4EBP1 (Thr70) (Cell Signaling, USA) at dilutions of 1:100 overnight at 4°C. Detection was with a Catalyzed Signal Amplification Kit (DAKO Co, Carpinteria, USA) and visualization was with 3, 3'-diaminobenzidine (DAB).
IHC results were evaluated and scored independently by two pathologists without knowledge of patient clinicopathological outcomes. IHC expression levels for LMP1, p-mTOR, p-P70S6K and p-4EBP1 were assessed by a semi-quantitative scoring system according to the intensity of staining and percentage of tumor cells stained. Staining intensity was scored as 0 = negative, 1 = weak, 2 = moderate, 3 = strong. The percentage of tumor cells stained was scored as 0 = no tumor cells stained, 1 = 1-10% of tumor cells stained, 2 = 11-50% of tumor cells stained, 3 = 51-100% of tumor cells stained. The two individual parameters were added, resulting in an immunoreactivity score (IRS) ranging from 0 to 6. We defined cases with IRS ≥ 4 as high expression, and cases with IRS < 4 as low expression.
Cell culture and plasmids
The EBV-negative human NPC cell lines HONE1 and 6-10B, and the EBV-positive NPC cell line C666-1 were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 units of penicillin/ml and 100 μg of streptomycin/ml. All cells were maintained in a humidified incubator at 37°C with 5% CO2.
The eukaryotic expression plasmid pZipNeoSV-LMP1 containing the B95.8-LMP1 gene was kindly provided by Professor Kai-Tai Yao from Nan Fang Medical University (Guangzhou, China) .
Transient and stable transfection
Briefly, 4 × 105 cells per well were plated into six-well plates and grown for one day in antibiotic-free medium containing 10% FBS prior to transfection. Plasmid pZipNeoSV-LMP1 and control vector transfection were performed with Lipofectamine 2000 (Invitrogen, CA) according to the manufacturer's instructions. Further assays were conducted after 48 h incubation of transiently transfected cells.
To generate the stable transfected cell lines HONE1-LMP1 and HONE1-vector, cells were passaged at 1:6 into fresh growth medium 24 h after transfection. G418 (Amresco, USA) at a final concentration of 150 μg/ml was added to complete medium to select resistant cells. Clones were separated and expanded into stable cell lines.
Western blot analysis
Transfected cells were harvested and lysed with RIPA buffer (Upstate, USA). Denatured proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes (Roche, Germany), and incubated with primary antibodies LMP1 (BD, USA), p-IκBα, phosphatase and tensin homolog (PTEN), Poly ADP-ribose polymerase (PARP), Survivin, AKT1, p-AKT (Thr308) (Santa Cruz, USA), mTOR, p-mTOR(Ser2448), p-P70S6K(Thr389), p-4EBP1 (Thr70) (cell signaling, USA) and p-NF-κB p65 (Ser276) (Kangchen, China) overnight at 4°C in 5% skimmed milk/TBST (Tris-buffered saline solution containing 0.1% Tween 20) at a dilution of 1:1000. GAPDH (1:3000 dilution, Santa Cruz, USA) was used as internal control. Horseradish peroxidase-conjugated second antibody incubation was followed by chemiluminescence detection with an ECL Western blot Kit (Cell Signaling Technology, USA). Densitometry to quantify proteins was conducted by Image J 1.37 v software (NIH, USA).
Total RNA was isolated by Trizol (Invitrogen, CA) and purified by Nucleospin RNA clean-up (MN, USA). All procedures were performed according to the manufacturer's instructions. Formaldehyde agarose gel electrophoresis was carried out to quantify the total RNA.
cDNA microarray analysis
The human 22 K oligonucleotide microarray comprised 21,329 probes from the Operon Company (Human Genome Oligo Set Version 2.1), constructed by CapitolBio Corporation (Beijing, China). Hybridization to each array was performed with equivalent amounts of HONE1-LMP1 and control HONE1-vector samples that were differentially fluorescence-labeled with Cy3 or Cy5. Fluorescence exchange experiments were performed. Hybridization and image capture were as previously described . Normalization was based on a LOWESS program . All original data was submitted to the Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/projects/geo/ with the accession number GSM467646. Genes with signal intensity (Cy3 or Cy5) > 800 were regarded as expressed, and alteration ratios above 1.3-fold, or lower than 0.7, were defined as differential expression. Pathways analysis of all differentially expressed genes was performed according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
Quantitative real-time PCR (Q-RT-PCR)
To validate the microarray results, five genes associated with the mTOR signaling pathway were analyzed by Q-RT-PCR. Primers were designed by Primer 5.0 (Additional file 1). Following the manufacturer's protocols, cDNA was prepared from 2 μg total RNA by M-MLV reverse transcriptase (Promega, USA) and amplified with a DNA Master SYBR Green I Kit (Roche, Germany). The relative expression ratio was determined by the formula 2-ΔΔCt (ΔΔCt = ΔCtHONE1-LMP1-ΔCtHONE1-Vector, ΔCt = Ctgene-CtGAPDH, where Ct is the cycle number at which the fluorescence signal exceeds background) .
Small interfering RNA (siRNA) transfection
The LMP1 and negative control siRNA were chemically synthesized by GenePharma Corporation (Shanghai, China). The sequences of LMP1 siRNA (EU000388, miRNA nucleotide 371-389) were: sense sequence, 5'-GGA AUU UGC ACG GAC AGG CTT-3'; anti-sense sequence, 5'-GCC UGU CCG UGC AAA UUC CTT-3' [19, 20]. The sequences of negative control siRNA were: sense sequence, 5'-UUC UCC GAA CGU GUC ACG UTT-3'; anti-sense sequence, 5'-ACG UGA CAC GUU CGG AGA ATT-3'. The EBV-positive NPC cell line C666-1 was seeded in a 24-well plate with 4×104 cells per well in growth medium without antibiotics the day before transfection. Following the manufacturer's instruction, 1 μl Lipofectamine2000 was used in each well with final siRNA concentration at 50 nM or 100 nM.
After 72 h of siRNA transfection, cells were harvested and washed thrice with PBS, suspended in PBS and centrifuged on the slides. Slides were fixed with 4% paraformaldehyde for 30 min, permeabilized, and cells covered with 0.1% Triton X-100 for 15 min. After 1 h blocking in PBS + 0.1% Tween plus 1% bovine serum albumin, cells were incubated with primary antibodies of LMP1 (BD, USA), p-mTOR (Ser2448) and p-4EBP1 (Thr70) (Cell Signaling, USA) at 4°C overnight, then with secondary antibody for 1 h at room temperature. After counterstaining with DAPI (1 μg/ml) for 10 min, slides were observed and photographed with confocal microscopy.
Data was analyzed using SPSS16.0 software (SPSS Inc., Chicago, USA). The correlation between LMP1, p-mTOR, p-P70S6K, p-4EBP1 expression and clinicopathological parameters was assessed by chi-square test. The correlation between LMP1 and p-mTOR, p-P70S6K, p-4EBP1 expression was measured by Spearman's correlation test. Kaplan-Meier analysis and log-rank test were used to assess survival rate and compare survival rate differences. Univariate and multivariate regression analysis were performed with the Cox proportional hazards regression model to analyze the factors related to prognosis. A p-value less than 0.05 was considered statistically significant.