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To explore the role of endothelin-1 (ET-1) gene in regulating the proliferation, migration and invasion of nasopharyngeal carcinoma cells.A lentivirus-mediated shRNA-ET-1 vector was infected into 5-8F cells, and the interference efficiency was examined with Western blotting. MTT assay, cell cycle analysis, plate colony formation assay, Transwell assay, Boyden chamber assay and tumor growth assay were carried out to analyze the changes in cell proliferation, migration and invasion. The expressions of genes related with epithelial-mesenchymal transition (EMT) were examined using Western blotting.shRNA-ET-1 transfection significantly inhibited the expression of ET-1, and suppressed the growth, migration and invasion of 5-8F cells. ET-1 knockdown enhanced the expression of E-cadherin and CK18 and inhibited the expression of N-cadherin and vimentin.ET-1 promotes cell growth, migration and invasion by modulating the genes associated with epithelial-mesenchymal transition in nasopharyngeal carcinoma cells.

To study the changes in the intensity and temporal pattern of target gene expression in the tumor tissue of nude mice bearing human nasopharyngeal carcinoma (NPC) following injection of recombinant adeno-associated virus (rAAV) and recombinant adenovirus (AdV) in vivo.EBV-positive human NPC cell line C666-1 was inoculated subcutaneouly in nude mice. After the tumor mass reached 3 mm in diameter, 1.5 × 10(11) v.g (virus genome) rAAV-EGFP, 2.5 × 10(8) pfu rAdV-EGFP or their balanced mixture was injected intratumorally. At 5 and 10 days after the injection, the tumor tissues were harvested for immunohistochemical staining of GFP, and the ratio of the GFP-positive cells and the intensity of GFP expression was determined.Immunohistochemistry for GFP showed that 5 days after the injection, GFP expression was detected (1.70 ∓ 0.48) in the tumor tissue in rAAV group, and the peak expression levels was seen in rAdV group (6.00∓1.94); the expression level was comparable between the combination group (6.90 ∓ 1.92) and rAdV group. At 10 days, GFP expression was considerably lowered to 2.00 ∓ 0.67 in rAdV group but increased to 8.00∓1.15 in rAAV group. The expression in the combination group maintained a high level at 10 days (10.10∓1.63), which was significantly higher than that in rAAV group (P%0.001).Transfection with rAAV combined with rAdV allows instant, sustained and significantly enhanced expression of the target gene in the tumor tissue. This approach takes advantages of the two viruses and can be ideal for exogenous gene delivery into the tumor tissues.

To detect the serum proteomic fingerprints in patients with hypopharyngeal squamous cell carcinoma (HPSCC) by surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) protein chip array technique.The serum samples were obtained from 58 HPSCC patients for protein expression analysis using SELDI-TOF Protein Chip technique and cation-exchange (CM10) protein array. All the spectra were compared and the qualified mass peaks with mass-to-charge ratios (m/z) between 1 and 70 kD were autotimatically detected. The tree analysis pattern was generated using Biomarker Patterns Software.The protein profiles of HPSCC serum were analyzed according to the clinical and pathological features of the patients and their treatment response. No significant difference was noted in the serum proteins between HPSCC patients with different statuses of cervical lympha node metastasis (P0.05), and the difference between well differentiated and poorly differentiated HPSCC was only minor. No significant difference was found in the serum proteins between chemotherapy-sensitive patients and the insensitive patients (P0.05), but 5 proteins were identified to be overexpressed in the sensitive patients (P/ = 0.05). Radiotherapy-sensitive HPSCC patients were segregated from the insensitive group with a sensitivity of 86.67% and specificity of 100%.The serum protein at the m/z value of 6115.74 is overexpressed in radiotherapy-sensitive HPSCC patients. Serum protein profiling allows the prediction of radiotherapy response in HPSCC patients, and the identified proteins may serve as candidate biomarkers for predicting the radiotherapy sensitivity of HPSCC.

To construct a miR-155-based lentivirus vector to induce cyclooxygenase-2 gene silencing in nasopharyngeal carcinoma (NPC) cells by expressing anti-COX-2 shRNAmir.miR-155-based anti-COX-2 shRNAmir template was synthesized and inserted into pLVTHM plasmid. The recombinant pLVTHM/shRNAmir was transfected into 293FT cells for packaging the lentivirus vector. After infection with the lentivirus vector, the GFP-positive cells were screened by flow cytometry, and COX-2 mRNA level was detected by RT-PCR.Restriction digestion and DNA sequencing confirmed successful construction of the anti-COX-2 vector pLVTHM/shRNAmir. A subline of C666-1 cells was established after infection with the lentivirus vector, and the COX-2 expression in the cells was stably silenced.The shRNAmir lentivirus vector constructed may serve as an effective COX-2 inhibitor, which may facilitate future studies of gene therapy of NPC.

To study the role of Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) in nasopharyngeal carcinoma (NPC) cell growth and metastasis in vivo by recombinant adeno-associated virus (rAAV)-mediated RNA interference (RNAi).Specific small hairpin RNA (shRNA) targeting EBV-LMP-1 gene was designed and synthesized to construct two rAAV vectors rAAV-shRNA-LMP-1 and rAAV-EGFP. The multiplicity of infection (MOI) was confirmed using different titers of rAAV-EGFP to transfect the NPC cell line C666-1. The C666-1 cells were transfected by rAAV-shRNA-LMP-1 at the optimal MOI titer and the inhibition efficiency of the target gene expression was determined with RT-PCR. The C666-1 cells with RNAi of LMP-1 gene were injected into nude mouse liver via laparotomy to establish the animal model of hepatic and lung metastases of NPC cells. The metastases of the C666-1 cells in the liver and lungs were observed to assess the effect of LMP-1 gene silencing on the tumorigenic and metastatic potentials of the cells in vivo.The transfection efficiency of 5 x 10(4) virus genome/cell rAAV-EGFP exceeded 95%. The expression of the target gene was suppressed by over 90% as shown by RT-PCR after transfection with rAAV-shRNA-LMP-1 at 5 x 10(4) virus genome/cell. Animal experiments showed that compared with rAAV-EGFP, rAAV-shRNA-LMP-1 transfection did not reduce the primary tumor volume implanted into the liver, but significantly inhibited the intrahepatic and lung metastases of the NPC cells.LMP-1 expression can be suppressed effectively by rAAV-mediated RNAi, and LMP-1 suppression does not obviously affect the tumor cell growth but can inhibit their metastasis in vivo.