Epstein / 中南大学学报(医学版)

Epstein-Barr virus (EBV), a definite tumorigenic virus, is closely related to the development of nasopharyngeal cancer, gastric cancer, lymphoma and other tumors. EBV encodes a total of 44 mature microRNAs, which can regulate the expression of virus and host genes. EBV-encoded microRNAs and their regulated target molecules participate in the biological functions of tumor apoptosis, proliferation, invasion, and metastasis during tumorigenesis and development, and play an important role in the development of tumor.

miR-18a enhances the radiosensitivity of nasopharyngeal carcinoma cells through inducing autophagy / 中华耳鼻咽喉头颈外科杂志

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.

MicroRNA-424-5p inhibits the proliferation, migration, and invasion of nasopharyngeal carcinoma cells by decreasing AKT3 expression

This study examined the expression and potential mechanism of microRNA (miRNA)-424-5p in nasopharyngeal carcinoma (NPC). NPC tissues were collected from 40 patients who were enrolled in the study, and skin samples were collected from 26 healthy subjects during plastic surgery as controls. We performed various in vitro assays using miR-424-5p to examine its function in primary NPC-1 cells. Bioinformatics was employed to analyze potential target genes and signaling pathways of miR-424-5p. We found that miR-424-5p expression in NPC tissues is downregulated and negatively correlated with lymph node metastasis and clinical staging. Expression of miR-424-5p in NPC cells was also downregulated, and transfection with miR-424-5p mimics inhibited proliferation, migration, and invasion of NPC-1 cells. Bioinformatics identified the AKT3 gene as a potential target of miR-424-5p and dual luciferase assays confirmed this finding. Upregulation of AKT3 expression rescued the inhibitory effect of miR-424-5p on the proliferation, migration, and invasion. Our results suggest that miR-424-5p inhibited the proliferation, migration, and invasion of NPC cells by decreasing AKT3 expression.

Effects of DACT1 methylation status on invasion and metastasis of nasopharyngeal carcinoma

BACKGROUND: The purpose of the present study was to investigate the role of the methylation status of the DACT1 gene on the invasion and metastasis of nasopharyngeal carcinoma cells. METHODS: The levels of methylation and expression of the DACT1 gene in nasopharyngeal carcinoma tissues and CNE2 cells were determined by methylation-specific PCR and RT-PCR, respectively. CNE2 cells were treated with 5-aza-2-deoxycytidine, and the variation in the methylation status of the DACT1 gene was detected, as well as the influence of methylation on invasiveness of nasopharyngeal carcinoma cells. RESULTS: The DACT1 gene was hyper-methylated in 44 of 62 cases of nasopharyngeal carcinoma. The DACT1 gene was hyper-methylated in 32 of 38 cases of nasopharyngeal carcinoma with lymph node metastasis, and the DACT1 gene was hyper-methylated in 7 of 24 cases of nasopharyngeal carcinoma without lymph node metastasis. The DACT1 mRNA level was weakly expressed or not expressed in all nasopharyngeal carcinoma tissues with hyper-methylated DACT1 genes; however, the DACT1 mRNA level was highly expressed in nasopharyngeal carcinoma tissues with low expression of the methylated DACT1 gene. The DACT1 gene was hyper-methylated and not expressed in CNE2 cells that did not have 5-aza-2-deoxycytidine treatment. After 5-aza-2-deoxycytidine treatment, the DACT1 gene was demethylated and the expression of DACT1 was restored. Moreover, the invasion ability was inhibited in CNE2 cells treated with 5-aza-2-deoxycytidine. CONCLUSION: The expression of DACT1 was related to the methylation status. High expression of DACT1 may inhibit the invasion and metastasis of nasopharyngeal carcinoma cells.

miR-141 is up-regulated in biopsies from Vietnamese patients with nasopharyngeal carcinoma

Abstract: Novel biomarkers for screening, diagnosis and monitoring the treatment of nasopharyngeal carcinoma (NPC), one of the most common cancers in Vietnam, are urgently required. Increasing evidence suggests that microRNA-141 (miR-141) is associated with NPC, owing to its ability to affect the expression of genes that modulate tumorigenesis. Unfortunately, research on miR-141 expression in Vietnamese patients is limited. Therefore, the objective of the current study was to evaluate miR-141 expression and assess whether miR-141 might be a potential biomarker for diagnosis of NPC in Vietnamese patients. Total RNA isolated from 40 NPC biopsy samples and 37 non-cancerous samples was analyzed by quantitative reverse-transcription PCR. The miR-141 expression levels were compared between NPC biopsy and non-cancerous samples. The frequency of miR-141 detection was 37.50% and 10.80% in the NPC and non-cancerous samples, respectively (p = 0.0143). The miR-141 expression was 5.27 times higher in tumor samples than non-cancerous samples. Additionally, the RR (Relative risk) and OR (Odds ratio) were 1.83 (95%CI = 1.2576-2.6675, p = 0.0016) and 4.95 (95%CI = 1.4625-16.7541, p = 0.01), respectively. In conclusion, miR-141 was up-regulated in the biopsy samples and thus may be a potential biomarker for NPC in the Vietnamese population.

Identification of key genes involved in nasopharyngeal carcinoma / Identificação dos principais genes envolvidos no carcinoma nasofaríngeo

Abstract Introduction: Nasopharyngeal carcinoma is the most common cancer originating from the nasopharynx. Objective: To study the mechanisms of nasopharyngeal carcinoma, we analyzed GSE12452 microarray data. Methods: GSE12452 was downloaded from the Gene Expression Omnibus database and included 31 nasopharyngeal carcinoma samples and 10 normal nasopharyngeal tissue samples. The differentially expressed genes were screened by ANOVA in the PGS package. Using the BiNGO plugin in Cytoscape and pathway enrichment analysis in the PGS package, functional and pathway enrichment analyses were performed separately to predict potential functions of the differentially expressed genes. Furthermore, Transcription factor-differentially expressed gene pairs were searched, and then the transcription factor-differentially expressed gene regulatory network was visualized using Cytoscape software. Results: A total of 487 genes were screened as differentially expressed genes between the nasopharyngeal carcinoma samples and the normal nasopharyngeal tissue samples. Enrichment analysis indicated that PTGS2 was involved in the regulation of biological process and small cell lung cancer. ZIC2 and OVOL1 may function in nasopharyngeal carcinoma through targeting significantly up-regulated genes (such as PTGS2, FN1, CXCL9 and CXCL10) in the Transcription factor-differentially expressed gene regulatory network (e.g., ZIC2→PTGS2 and OVOL1→CXCL10). Conclusion: PTGS2, FN1, CXCL9, CXCL10, ZIC2 and OVOL1 might play roles in nasopharyngeal carcinoma.

Resumo Introdução: O carcinoma nasofaríngeo é o câncer mais comum originário da nasofaringe. Objetivo: Estudar os mecanismos do câncer de nasofaringe; dados do microarray GSE12452 foram analisados. Método: GSE12452 foi obtido da base de dados Gene Expression Omnibus e inclui 31 amostras de carcinoma nasofaríngeo e 10 amostras de tecido nasofaríngeo normal. Os genes diferencialmente expressos foram analisados por ANOVA no kit PGS. Usando o plugin BiNGO no Cytoscape e análise de enriquecimento da via no kit PGS, análises de enriquecimento funcional e da via foram realizadas separadamente para prever as potenciais funções dos genes diferencialmente expressos. Além disso, os pares Fator de Transcrição - genes diferencialmente expressos foram pesquisados e em seguida a sua rede reguladora foi visualizada usando o programa Cytoscape. Resultados: Um total de 487 genes foram analisados como genes diferencialmente expressos entre as amostras de carcinoma nasofaríngeo e amostras de tecido nasofaríngeo normal. A análise de enriquecimento indicou que PTGS2 estava envolvido na regulação do processo biológico e câncer pulmonar de pequenas células. ZIC2 e OVOL1 podem funcionar no carcinoma nasofaríngeo almejando-se de maneira significativa os genes suprarregulados (como o PTGS2, FN1, CXCL9 e CXCL10) na rede reguladora de fator de transcrição - genes diferencialmente expressos (p.ex., ZIC2→PTGS2 e OVOL1→CXCL10). Conclusão: PTGS2, FN1, CXCL9, CXCL10, ZIC2 e OVOL1 podem desempenhar alguns papéis no carcinoma de nasofaringe.

Excavation of attractor modules for nasopharyngeal carcinoma via integrating systemic module inference with attract method

The molecular mechanism of nasopharyngeal carcinoma (NPC) is poorly understood and effective therapeutic approaches are needed. This research aimed to excavate the attractor modules involved in the progression of NPC and provide further understanding of the underlying mechanism of NPC. Based on the gene expression data of NPC, two specific protein-protein interaction networks for NPC and control conditions were re-weighted using Pearson correlation coefficient. Then, a systematic tracking of candidate modules was conducted on the re-weighted networks via cliques algorithm, and a total of 19 and 38 modules were separately identified from NPC and control networks, respectively. Among them, 8 pairs of modules with similar gene composition were selected, and 2 attractor modules were identified via the attract method. Functional analysis indicated that these two attractor modules participate in one common bioprocess of cell division. Based on the strategy of integrating systemic module inference with the attract method, we successfully identified 2 attractor modules. These attractor modules might play important roles in the molecular pathogenesis of NPC via affecting the bioprocess of cell division in a conjunct way. Further research is needed to explore the correlations between cell division and NPC.

Role of regenerating gene IA expression on local invasion and survival in nasopharyngeal carcinoma

BACKGROUND: Regenerating gene IA (REGIA) plays an important role in tissue regeneration and tumors prognosis of epithelium origin. However, the role of REGIA in nasopharyngeal carcinoma (NPC) is unclear. This study aims to investigate the expression and function of REG1A in NPC. RESULTS: We have found that there was 63 patients with REGIA positive expression of 155 patients in this study (40.65%). The positive expression rate of REGIA was 30.50, 44.44 and 47.83% in stage T2, T3 and T4 patients, respectively. The REGIA expression was significantly difference in T2 and T4 stage tumors or T2 and T3-T4 stage. The positive expression rate of REGIA was found to be higher in patients with cervical lymph node persistence than those with cervical lymph node complete regression. Patients with negative REGIA expression had a better overall survival and free survival than those with REGIA positive expression. In addition, according to the univariate and multivariate analysis, the REGIA expression was an independent adverse prognostic factor for NPC patients. CONCLUSION: REGIA expression was a useful biomarker in NPC patients for assessing T stage and survival.

Real-time PCR assay for rapid detection of GSTM1 polymorphism in nasopharyngeal carcinoma patients.

Nasopharyngeal carcinoma (NPC) is a common public health problem in Thailand. Glutathione S-transferase M1 gene deletion (GSTM1 null genotype) carriers have been reported to be at increased risk and therefore this parameter is a potential marker for screening of NPC high-risk individuals. However, the conventional polymerase chain reaction (C-PCR) assay commonly used for GSTM1 null genotype detection is not suitable for mass screening since it is inconvenient, time consuming and unsafe due to the use of a toxic chemical. Currently, real-time PCR (R-PCR) assay is recommended for quicker and safer detection of various genetic polymorphisms. The aim of this study was to develop a SYBR green I R-PCR assay combined with melting curve analysis for GSTM1 polymorphism detection in Thai NPC patients. The results were compared to those from the C-PCR assay using DNA samples from peripheral blood leukocytes of 120 Thai NPC patients. The frequencies of GSTM1 polymorphism detected by the R-PCR and the C-PCR were the same. Forty-eight individuals that were GSTM1+ in the R-PCR assay showed 2 peaks with melting points of 82.5 and 87.5 that correlated with the appearance of 2 DNA bands in the C-PCR assay (i.e., one for GSTM1 at 215 base pairs (bp) and one for ?-globin at 268 bp). By contrast, 72 individuals that were GSTM1?- in the R-PCR assay showed 1 peak with a melting point of 87.5C that correlated with the appearance of 1 DNA band for -globin at 268 bp in the C-PCR assay. The R-PCR assay using SYBR Green I and melting curve analysis for GSTM1 polymorphism detection was as reliable as C-PCR assay but was quicker and safer and more amenable to large scale screening in Thai NPC cases.

Expresión inmunohistoquímica de metaloproteasas (MMP-1, 2 y 11) e inhibidor de metaloproteasas de tejido-1 (TIMP-1), y expresión de p53 en linfomas angiocéntricos de células T/NK tipo nasal / Metalloproteinase (MMP-1, 2 and 11), tissue inhibitor of metalloproteinase-1 (TIMP-1), and p53 expression in nasal-type angiocentric T/NK-cell lymphoma. An immunohistochemical study

Se analizan 20 casos de linfomas extraganglionares de células T/NK de tipo nasal, estudiados en el Instituto Nacional de Cancerología, México, D. F., para su expresión inmunohistoquímica de las células neoplásicas, expresión nuclear de la proteína supresora de tumor p53, así como de enzimas que participan en invasión, destrucción tisular y metástasis: metaloproteasas. Material y métodos: Se estudió el material quirúrgico de estos casos y se efectuó tinción con hematoxilina y eosina analizando sus características histopatológicas: tamaño celular y detalle citológico. Se realizó estudio de inmunohistoquímica para corroborar el tipo celular, así como CD3 (células T), CD56 (células NK), expresión nuclear de la proteína supresora de tumor p53, y la expresión de metaloproteasas tipo 1, 2, 11 (MMP-1, 2, 11) y un inhibidor de metaloproteasas 1 (TIMP-1). Se analizaron variables demográficas, como edad del paciente, sexo, localización del tumor primario, etapa clínica, tratamiento en general y seguimiento. Estudio estadístico: Se analizó la prueba exacta de Fisher para correlacionar la expresión entre las metaloproteasas y su diferencial entre las células epiteliales, tumorales, estromales, necrosis y células endoteliales. Resultados: Los 20 casos fueron positivos CD3 citoplásmico, CD56, 19 de ellos positivos a p53, cinco de ellos con positividad nuclear mayor al 50% de las células neoplásicas. Hubo una mayor expresión citoplásmica tumoral de MMP-1; mayor expresión citoplásmica en el epitelio de TIMP1 y MMP-11. Los pacientes con sobreexpresión de p53 tuvieron un curso clínico fatal. Tres de ellos recibieron únicamente radioterapia falleciendo dentro del primer mes del tratamiento. Discusión: Los linfomas angiocéntricos de células T/NK tipo nasal son neoplasias frecuentes en los países de Asia, Latinoamérica, incluyendo a México. Frecuentemente esta patología se asocia a VEB con expresión fenotípica de células T/NK, cuyas características histológicas son: atipia celular linfoide, angioinvasión y necrosis, reflejado en los pacientes con destrucción progresiva de los tejidos blandos del macizo facial y curso clínico fatal.

Twenty cases of extraganglionar Nasal type T/NK cell lymphomas were analyzed at the National Cancer Institute of Mexico. We studied immunophenotype of neoplastic cells, nuclear p53 expression, and enzymes as matrix metalloproteinases participating in invasion, tissular destruction and metastases. Material and Methods: Paraffin blocks from all cases were retrieved and analyzed by hematoxilin and eosin. Histopathological features included cellular size and cytologic characteristics. We performed immunohisto chemistry to determine CD3, CD56, p53 cellular type and expression of (MMPs-1, 2,11) matrix metalloproteinases and one tissue inhibitor of TIMP 1 metalloproteinase. Demographic variables included, age, sex, primary location, clinical stage, treatment and follow up. Statistical analysis: The association of different matrix metalloproteinases in epithelial and tumoral cells, stroma, necrosis and endothelial cells were found to be significant using Fisher s exact test. Results: All studied cases were positive to cytoplasmic CD3, CD56 (NK cells), 19 of them were positive to p53, five of them with nuclear overexpression of p53 in more than 50% of neoplastic cells. There was significant expression of MMP-1 in tumoral cells; the epithelium displayed significant expression of TIMP 1 and MMP-11. Patients with p53 overexpression displayed a poorer prognosis. Three of them had undergone radiotherapy and died within the first month of treatment. Discussion: This type of lymphoma is a common neoplasm in Asia, Latin America and Mexico. It is worth noting it has has been linked to Epstein Barr virus with T/NK-cell phenotype, which often displays cellular atypia, an angiocentric growth pattern and necrosis. It is clinically expressed by progressive destruction of midline facial soft tissue and has a poor prognosis.

Genetic alterations in Ki-ras and Ha-ras genes in juvenile nasopharyngeal angiofibromas and head and neck cancer

Context: Ras gene mutations have been associated to a wide range of human solid tumors. Members of the ras gene family (Ki-ras, Ha-ras and N-ras) are structurally related and code for a protein (p21) known to play an important role in the regulation of normal signal transduction and cell growth. The frequency of ras mutations is different from one type of tumor to another, suggesting that point mutations might be carcinogen-specific. Objectives: To study the occurrence of Ki-ras and Ha-ras mutations. We also studied the relative level of Ha-ras mRNA in 32 of the head and neck tumors. Design: Case series. Setting: University referral unit. Participants: 60 head and neck tumors and in 28 Juvenile Nasopharyngeal Angiofibromas (JNA). Diagnostic test: Using PCR-SSCP we examined the occurence of Ki-ras and Ha-ras mutations. The relative level of Ha-ras mRNA was examined by Northern blot analysis. Results: None of the head and neck tumors or JNA samples showed evidence of mutations within codons 12,13,59 and 61 of Ki-ras or Ha-ras genes. However, 17 (53 per cent) of the tumors where gene expression could be examined exhibited increased levels of Ha-ras mRNA compared with the normal tissue derived from the same patient. Conclusions: Our results demonstrate for the first time that mutations of Ki-ras and Ha-ras genes are not associated with the development of JNA and confirm previous reports indicating that activating ras mutations are absent or rarely invloved in head and neck tumors from western world patients. Furthermore, our findings suggest that overexpression of Ha-ras, rather than mutations, might be an important factor in the development and progression of head and neck tumors.

Microsatellite instability in Epstein-Barr virus associated with nasopharyngeal carcinoma.

The result reported here represents the first human genomic screen for MSI in Epstein-Barr-Virus associated NPC. The analysis revealed the incidence of MSI only 1 of 23 cases (4%) which indicates that MSI is less common in NPC development.

Loss of heterozygosity for chromosome 11 in Epstein-Barr-virus associated nasopharyngeal carcinoma.

In order to demonstrate and define possible tumor suppressor gene loci on chromosome 11 associated with NPC, we used 7 STR to test for LOH on 25 NPC samples. LOH was detected in 46 per cent of cases. Most LOH loci were clustered on the long arm. Further study demonstrated 22 per cent and 45.5 per cent of cases with LOH on 11q13 and 11q23 respectively.

Cytogenetic and viral studies in nasopharyngeal carcinomas in patients of Indian origin.

Four cases of undifferentiated nasopharyngeal carcinomas (NPC) (grade III-IV) in patients of Indian origin were investigated for specific chromosome markers and evidence of Epstein-Barr virus (EBV) positivity. Abnormalities involving chromosome #3, like del (3) (p24-pter) and 3q+(q27-qter) were found in these patients, similar to earlier reports in patients of Chinese and Kenyan origin2,4,13 who however were EBV positive, unlike the patients in this study who were EBV negative. Implications of the cytogenetic and serological data in Indian patients with NPC, available for the first time, may throw some light on the etiology of the disease in this ethnic group where nasopharyngeal carcinoma is also endemic.