OBJECTIVE: To filtrate and prove the different microRNAs (miRs) profiles in nasopharyngeal carcinoma. METHOD: Screening the different expressions of miRs between nasopharyngeal carcinoma and the inflammatory tissues by the application of expression profiling of chip high-throughput and large-scale microarray analysis. Then we used RT-QPCR technology to prove the accuracy of screening results. RESULT: There were significant expression differences of miRs between nasopharyngeal carcinoma and the control tissues, 144 human miRs had 2 or more fold the difference ratio. Compared with the inflammatory tissues, we have found that miRs-34b, miRs-449b and miRs-7-1 significantly low expressed in nasopharyngeal carcinoma, yet miRs-125b, miRs-184, miRs-196b, miRs-205 and miRs-24-1 expressed high. The results were consistent with the microarray analysis. CONCLUSION The difference expressed miRs might be closely related to the process of nasopharyngeal carcinoma, and the research on miRs profiles maybe provide a powerful target basis for early diagnosis and therapy of nasopharyngeal carcinoma.
Carcinoma ; Gene Expression Profiling ; Humans ; MicroRNAs ; genetics ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics ; Oligonucleotide Array Sequence Analysis

Objective To screen out the potential prediction genes for nasopharyngeal carcinoma(NPC)from the gene microarray data of NPC samples and then verify the genes by cell experiments.Methods The NPC dataset was downloaded from Gene Expression Omnibus,and limma package was employed to screen out the differentially expressed genes.Weighted correlation network analysis package was used for weighted gene co-expression network analysis,and Venn diagram was drawn to find the common genes.The gene ontology annotation and Kyoto encyclopedia of genes and genomes pathway enrichment were then performed for the common genes.The biomarkers for NPC were further explored by protein-protein interaction network,LASSO regression,and non-parametric tests.Real-time quantitative PCR and Western blotting were employed to determine the mRNA and protein levels of key predictors of NPC,so as to verify the screening results.Results There were 622 up-regulated genes and 351 down-regulated genes in the GSE12452 dataset.A total of 116 common genes were obtained by limma analysis and weighted gene co-expression network analysis.The common genes were mainly involved in the biological processes of cell proliferation and regulation and regulation of intercellular adhesion.They were mainly enriched in Rap1,Ras,and tumor necrosis factor signaling pathways.Six key genes were screened out,encoding angiopoietin-2(ANGPT2),dual oxidase 2(DUOX2),coagulation factor Ⅲ(F3),interleukin-15(IL-15),lipocalin-2,and retinoic acid receptor-related orphan receptor B(RORB).Real-time quantitative PCR and Western blotting showed that the NPC cells had up-regulated mRNA and protein levels of ANGPT2 and IL-15 and down-regulated mRNA and protein levels of DUOX2,F3,and RORB,which was consistent with the results predicted by bioinformatics.Conclusion ANGPT2,DUOX2,F3,IL-15 and RORB are potential predictive molecular markers and therapeutic targets for NPC,which may be involved in Rap1,Ras,tumor necrosis factor and other signaling pathways.
Humans ; Nasopharyngeal Carcinoma/genetics* ; Interleukin-15 ; Dual Oxidases ; Computational Biology ; Nasopharyngeal Neoplasms/genetics*

Humans ; Carcinogenesis/genetics* ; Gene Expression Regulation, Neoplastic ; Genes, cdc ; Nasopharyngeal Carcinoma/genetics* ; Nasopharyngeal Neoplasms/genetics* ; RNA Helicases/metabolism*

OBJECTIVE: To construct nasopharyngeal carcinoma CNE-2 cell lines expressing stable fusion suicide gene CD/UPRT. UL49. METHOD: The plasmids of pcDNA3.1 (-)E6. BARF1p. CD/UPRT. UL49 was transfected into CNE-2 cells through lipofectamine, and the transfected CNE-2 cells were selected by G418 and prodrugs for getting the cells expressing fusion CD/UPRT. UL49 gene. The protein produced by the suicide gene was tested by Western-blotting in CNE-2 cells. RESULT: Suicide genes were expressed stably in CNE-2 cells. CONCLUSION We constructed nasopharyngeal carcinoma cell lines CNE-2 expressing stable suicide gene through lipofectamine.
Artificial Gene Fusion ; methods ; Carcinoma ; Cell Line, Tumor ; Genes, Transgenic, Suicide ; Humans ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics

OBJECTIVE: To examine the expression of the inhibitor alpha of nuclear transcription factor kappaB (IkappaBalpha) mRNA expression and its sequence characteristics in human nasopharyngeal carcinoma cell (NPC) lines CNE1, CNE2, HNE1 and HNE2. METHODS: Reverse transcription was performed with the total RNAs isolated from the NPC cell lines CNE1, CNE2, HNE1 and HNE2, as well as the transplanted tumor tissues with HNE1 cells. Then IkappaBalpha cDNA was amplified by PCR, and the products were used to examine IkappaB alpha mRNA expression and DNA sequencing, or the DNA sequencing after the products were cloned into plasmid vector. RESULTS: IkappaB alpha mRNA was expressed in all the 4 nasopharyngeal carcinoma cell lines. DNA sequencing showed that polymorphisms and 5 mutations (A825G, A975G, G576A, A655G and C653A) existed in IkappaB cDNA from the transplanted tumor tissues with HNE1 cells, CNE1 and CNE2 cells. CONCLUSION The expression of IkappaBalpha mRNA not only exists, but DNA polymorphisms and some additional mutations in IkappaBalpha cDNA are also detected in the nasopharyngeal carcinoma cells.
Base Sequence ; Carcinoma ; Cell Line, Tumor ; Humans ; I-kappa B Proteins ; genetics ; NF-KappaB Inhibitor alpha ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics ; Polymorphism, Genetic ; RNA, Messenger ; genetics

Objective: To explore the effect of dormant polyploid giant cancer cells (PGCC) on nasopharyngeal carcinoma (NPC) recurrence and to clarify the role of inhibition of autophagy in inhibiting NPC-PGCC formation and preventing NPC recurrence. Methods: NPC cells-derived PGCC (NPC-PGCC) were induced by paclitaxel (PTX), and the morphology, polyploid characteristics and cell activity of PGCC were identified by light microscopy, immunofluorescence and Live/Dead cell double staining assays. RNA-seq was used to analyze the differentially expressed genes between NPC-PGCC and diploid nasopharyngeal carcinoma cells CNE2. Functional enrichment and pathway annotation analysis of differentially expressed genes were performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG). The level of autophagy in NPC-PGCC cells was assessed by Western Blot and transmission electron microscopy analysis. The role of autophagy in the formation of NPC-PGCC and the effect of NPC-PGCC on the recurrence of nasopharyngeal carcinoma were studied using a highly clinically relevant mouse nasopharyngeal carcinoma recurrence model. Statistical analysis was performed using GraphPad Prism 6 and P-values<0.05 were considered statistically significant. Results: NPC-PGCC induced by paclitaxel had the characteristics of burst-like division after dormancy. GO enrichment and KEGG pathway analyses identified the significant biological processes and pathways mainly concentrated in autophagy and related pathways involving the differentially expressed genes between NPC-PGCC and diploid nasopharyngeal carcinoma cells CNE2. The autophagy level was significantly enhanced in NPC-PGCC cells. In a highly clinically relevant mouse nasopharyngeal carcinoma recurrence model, the number of PGCC in the primary tumor of the nude mice treated with cisplatin were higher than those of the other groups. In nude mice pretreated with autophagy inhibitor and then co-treatment with autophagy inhibitor and cisplatin, the number of PGCC in primary tumors was less and the recurrence rate was significantly lower than in other groups. Conclusions: The mechanism of dormant polyploid giant cancer cells formation is related to autophagy. Inhibition of autophagy can inhibit the formation of PGCC and thus prevent the recurrence of nasopharyngeal carcinoma.
Animals ; Autophagy ; Carcinoma/genetics* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Cisplatin/pharmacology* ; Gene Expression Regulation, Neoplastic ; Mice ; Mice, Nude ; Nasopharyngeal Carcinoma/genetics* ; Nasopharyngeal Neoplasms/pathology* ; Paclitaxel/pharmacology* ; Polyploidy

OBJECTIVETo establish a nasopharyngeal carcinoma (NPC) cell line CNE1-pLVTHM/BART7 with stable ebv-miR-BART7 overexpression.

METHODSThe recombinant lentivirus pLVTHM/BART7 expression plasmid was packaged into mature lentivirus by 293FT cells and used to infect CNE1 cells. Flow cytometry was employed for sorting the GFP(+) cells. The efficiency of ebv-miR-BART7 overexpression was determined using qRT-PCR.

RESULTSThe recombinant lentivirus plasmid pLVTHM/BART7 was successfully constructed and verified by PCR and sequencing. The expression of ebv-miR-BART7 in CNE1 cells infected with the lentivirus pLVTHM/BART7 was significantly increased as compared with the negative control and the blank control cells.

CONCLUSIONThe recombinant lentivirus vector pLVTHM/BART7 results in high and stable expression of ebv-miR-BART7 in infected CNE1 cells, which provides a useful cell model for further studies of the role of ebv-miR-BART7 in nasopharyngeal carcinoma.

Carcinoma ; Cell Line, Tumor ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; MicroRNAs ; Nasopharyngeal Neoplasms ; genetics ; Plasmids

OBJECTIVE: To investigate the expression and significance of miRNA-324-3p and its target gene WNT2B in tissue specimens of nasopharyngeal carcinoma (NPC) specimens. METHOD: qRT-PCR was used to detect the expression of miRNA-324-3p and WNT2B mRNA, and Western blot was applied to assay the expression of WNT2B protein in 39 cases of NPC specimens and 21 cases of non-carcinoma epithelium. The relationship between their expression levels and clinicopathological characteristics and their correlation with clinical pathological parameters was analyzed. RESULT: The expression of miRNA-324-3p was significantly down-regulated decreased but WNT2B mRNA/protein increased obviously in NPC specimens (P < 0.01). A negative correlation between miRNA-324-3p and WNT2B was spotted (P < 0.05). The expression levels of these markers were closely correlated with T stage, clinic stage and cervical lymph node metastasis (P < 0.05). CONCLUSION The loss of miRNA-324-3p and ectopic WNT2B might co-induce the initiation and progression of NPC.
Carcinoma ; Glycoproteins ; genetics ; metabolism ; Humans ; Lymphatic Metastasis ; MicroRNAs ; metabolism ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; metabolism ; Neoplasm Proteins ; metabolism ; RNA, Messenger ; metabolism ; Wnt Proteins ; genetics ; metabolism

OBJECTIVE: To investigate the association between-1304T/G polymorphism in the promoter of MKK4 gene and the susceptibility in sporadic nasopharyngeal carcinoma. METHOD: MKK4-1304T/G genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 90 NPC cases and 30 healthy controls. RESULT: The number of nasopharyngeal carcinoma patients carrying with TG+GG genotype was much higher than those of controls (82.2% vs 66.7%, χ² =10.076, P < 0.05). Analysis showed that compared with the-1304TT genotype, -1304TG heterozygous reduced risk of nasopharyngeal carcinoma 0.56 fold (95% CI = 0.164-1.178, P < 0.01) and-1304GG lower 0.58 fold (95% CI = 0.126-1.381, P < 0.01), TG+ GG genotype variation risk of nasopharyngeal carcinoma decreased 0.72 fold (95% CI = 0.105-0.753, P < 0.01). CONCLUSION MKK4 gene-1304TG genotype can reduce risk of nasopharyngeal carcinoma, and it may be an independent protection factor in sporadic nasopharyngeal carcinoma.
Carcinoma ; Genetic Predisposition to Disease ; Genotype ; Heterozygote ; Humans ; MAP Kinase Kinase 4 ; genetics ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Promoter Regions, Genetic

Objective: To explore the impacts of miR-18a overexpression or depression on the radiosensitivities of nasopharyngeal carcinoma cell line CNE1 and CNE2 and underlying mechanisms. Methods: CNE1 and CNE2 were transfected with miR-18a mimics, inhibitor and the corresponding control vectors. qRT-PCR and western blot were used to determine the ataxia telangiectasia mutated (ATM) expressions in CNE1 and CNE2. CNE1 and CNE2 with stably expressing miR-18a and miR-18a siRNA were constructed. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the impacts of the miR-18a overexpression or depression combined with irradiation on the cell growth. Flow cytometry was used to detect the cell apoptosis and cell cycle. Colony formation assay was used to evaluate the raodiosensitivities of cells. Acridine orange (AO) staining and western blot were used respectively to test the autophagy and the expressions of related proteins. Independent samples t test was used to compare the mean value between groups by using SPSS 16.0. Results: ATM mRNA was decreased significantly in CNE1 and CNE2 cells transfected with 100 or 200 nmol/L miR-18a mimics for 48 hours (CNE1: RQ=0.174±0.139 and 0.003±0.001, t=9.939 and 19 470.783;CNE2: RQ=0.024±0.008 and 0.019±0.012, t=270.230 and 137.746, respectively, all P<0.001). ATM proteins were also decreased after transfected with 100 or 200 nmol/L miR-18a mimics for 72 hours. While in the cells transfected with 100 and 200 nmol/L miR-18a inhibitor for 48 hours, the expressions of ATM mRNA were upregulated significantly (CNE1: RQ=9.419±2.495 and 2.500±1.063, t=-4.427 and -41.241; CNE2: RQ=7.210±0.171 and 115.875±15.805, t=-62.789 and -12.589, all P<0.05), and the expressions of ATM proteins increased after transfected for 72 hours. The growth of cells with miR-18a overexpression plus 4 Gy irradiation were obviously inhibited compared to that of cells with the 4Gy irradiation alone; while the growth of miR-18a-inhibited cells increased compared to that of cells with 4 Gy irradiation alone (all P<0.05). CNE1 transfected with 100 nmol/L miR-18a mimics plus 4 Gy irradiation showed the higher apoptosis rate than the cells with 4 Gy irradiation alone ((22.9±2.1)% vs. (16.3±1.0)%, t=-4.870, P<0.01). Compared to the cells with 4 Gy irradiation alone, miR-18a-overexpressed cells plus 4 Gy irradiation decreased their percentages in G1 phases ((20.2±3.0)% vs. (29.8±4.4)%, t=3.119) and G2/M phases ((21.5±0.9)% vs. (33.4±3.1)%, t=6.410, P<0.05 for both), and increased their percentages in S phases ((56.7±4.9)% vs. (36.8±6.4)%, t=-4.246, P<0.05), and these cells possessed less colony number after exposure to different doses of irradiation, more autophagy-lysosome number, and more expressions of LC3 proteins (all P<0.05). There were no significant differences in the expressions of p62 expressions between different groups of cells. Conclusion: Overexpression of miR-18a can enhance the radiosensitivities of NPC cells by targeting ATM to abrogate G1/S, G2/M arrest and to induce autophagy and apoptosis.
Apoptosis ; Autophagy ; Cell Line, Tumor ; Cell Proliferation ; G2 Phase Cell Cycle Checkpoints ; Humans ; MicroRNAs/genetics* ; Nasopharyngeal Carcinoma/genetics* ; Nasopharyngeal Neoplasms/genetics* ; Radiation Tolerance