AURKA, as a potential prognostic biomarker, regulates autophagy and immune infiltration in nasopharyngeal carcinoma.

BACKGROUND: Dysfunction of Aurora A (AURKA) plays crucial role in tumorigenesis and development of many types of cancer. However, the role of AURKA in nasopharyngeal carcinoma (NPC) has not been investigated yet. MATERIALS AND METHODS: Two independent NPC cohorts (GSE61218 and GSE102349) were enrolled from public database to investigate the expression level of AURKA between NPC and nasopharyngitis samples, the association of AURKA expression level with prognosis in NPC, and the potential mechanism of AURKA in NPC by using bioinformatics analyses. The expression level of AURKA protein in 62 paired NPC and nasopharyngitis tissues was evaluated by immunohistochemistry (IHC). Two NPC cell lines (SUNE-1 and CNE-2) were enrolled and the expression levels of AURKA in the NPC cells were inhibited by RNA interference. The expression levels of mRNAs were tested by qPCR and western-blotting. CCK-8 assay was applied to measure the cell growth. Cell migration was measured by using wound healing assays. RESULTS: AURKA was highly expressed in NPC samples compared to nasopharyngitis samples in GSE61218, which was confirmed by IHC. High expression of AURKA was associated with worse prognosis in GSE102349. Notably, silencing of AURKA was associated with significantly decreased cell growth and migration in NPC. Moreover, we found that the differentially expressed genes between high and low AURKA expression groups in GSE102349 were majorly enriched in both autophagy-related and immune-related pathways. Additionally, the expression level of AURKA was associated with the expression levels of autophagy-related genes and the infiltration of immune cells. CONCLUSION: AURKA overexpressed in NPC, which was associated with poor prognosis. Silencing of AURKA inhibited the proliferation and migration of NPC cells. Besides, AURKA might participate in the regulation of both autophagy and immunity in NPC.

SDF2L1 Inhibits Cell Proliferation, Migration, and Invasion in Nasopharyngeal Carcinoma.

The purpose of this study was to explore the relationship between stromal cell-derived factor 2-like 1 (SDF2L1) and nasopharyngeal carcinoma (NPC). 12 NPC tissues and 12 chronic nasopharyngitis tissues were involved in our study. Quantitative real-time PCR (qRT-PCR) and Western Blot were utilized to detect the expression of SDF2L1. Besides, immunofluorescence analysis was utilized to determine the protein expression of 97 paraffin-embedded NPC tissues and 58 nasopharyngitis tissues. Biological functional experiment included Cell Counting Kit-8 (CCK-8) assay, cell clone formation assay, cell scratch migration assay, Transwell migration assay, and Transwell invasion assay. All data were analyzed by SPSS. Results showed that downexpression of SDF2L1 was prominently present in NPC tissues and cells. Furthermore, silencing the expression of SDF2L1 promoted NPC proliferation, migration, and invasion in vitro, while overexpression of SDF2L1 has the opposite effect. In conclusion, SDF2L1 may act as a cancer suppressor gene, play a crucial role in the occurrence and development of NPC, and be a new therapeutic target or prognostic indicator for NPC.

Septin 9 Methylation in Nasopharyngeal Swabs: A Potential Minimally Invasive Biomarker for the Early Detection of Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is highly prevalent in Southeast Asia, and an unfavorable outcome is usually attributed to advanced stage NPC. Current methods for the early diagnosis of NPC have limitations in clinical practice. The aim of this study was to investigate the diagnostic ability of Septin 9 methylation for NPC. A quantitative methylation-sensitive PCR (qMS-PCR) assay was developed to measure the methylation status and levels of Septin 9 in nasopharyngeal tissues and paired swabs from patients with NPC, chronic nasopharyngitis, and healthy donors. Methylated Septin 9 was detected in 92% (23/25) of NPC tissues and 25% (4/16) of nasopharyngitis controls (p < 0.05). High-frequency hypermethylation with decreased mRNA expression of Septin 9 in NPC was also identified. Further, Septin 9 methylation was identified in 90.5% (19/21) of NPC biopsies and 71.4% (15/21) of paired swabs, indicating a good concordance between the two sample types. In addition, methylated Septin 9 was found in 16 (72.7%) nasal swabs from 22 NPC patients, 2 of 19 (10.5%) nasopharyngitis, but not in any of the healthy controls (p < 0.01). The methylation score in nasal swabs of the NPC group was also significantly higher than that of non-NPC controls (p < 0.001). Moreover, receiver operating characteristic (ROC) curve analysis showed an area under the curve (AUC) of 0.882 of Septin 9 methylation tests to discriminate NPC from non-NPC subjects. Our study demonstrated that frequent methylation of Septin 9 was present in NPC. Its detection in nasopharyngeal swabs may provide a minimally invasive and informative method for identifying early NPC cases.

Distinctive microRNA expression in early stage nasopharyngeal carcinoma patients.

The goal of this study was to investigate microRNAs (miRs) expression at different stages of nasopharyngeal carcinoma (NPC). MiR expression profiling at various stages of NPC was performed by miR array and further verified using quantitative real-time RT-PCR. Pathway enrichment analysis was carried out to identify the functional pathways regulated by the miRs. The expression of a selected group of identified miRs was verified in stage I NPC by in situ hybridization (ISH). A total of 449 miRs were identified with significantly different expressions between NPC tissues and normal pharyngeal tissues. Eighty-four miRs were dysregulated only in stage I NPC, among which 45 miRs were up-regulated and the other 39 were down-regulated. Pathway enrichment assay revleaed that three significantly down-regulated and three significantly up-regulated miRs involved in 12 pathways associating with tumour formation and progression. Quantitative RT-PCR confirmed the miR array result. In addition, the low expression levels of hsa-miR-4324, hsa-miR-203a and hsa-miR-199b-5p were further validated in stage I NPC by ISH. This present study identifed the miR signature in stage I NPC, providing the basis for early detection and treatment of NPC.

FCN2 c.772G>T polymorphism is associated with chronic adenoiditis and/or tonsillitis, but not -4 A>G and -602 G>A.

OBJECTIVE: Ficolins are complement activating peptides that play a role in the initial host defense against infectious pathogens. In the present study, we investigated the relationship between single nucleotide polymorphisms (SNPs) in the ficolin 2 gene (FCN2) and chronic adenotonsillitis in pediatric cases. STUDY DESIGN: Case-control study. METHODS: A total of 101 pediatric patients diagnosed with chronic adenotonsillitis and 100 healthy children were enrolled in the study. Genotypes of FCN2 promoter SNPs - 602 G>A and -4 A>G, and the exonic SNP c.772G>T were determined by light SNP assay after realtime PCR analysis using genomic DNA samples obtained from peripheral blood samples of all participants. RESULTS: Of the 101 chronic tonsillitis patients, 38 were girls and 63 were boys; the mean age was 5.2 ± 2.3 years. The c.772G>T SNP frequency was significantly higher in chronic adenotonsillitis cases compared to the control group (p = 0.00); however, no significant difference was determined at positions -602 G>A or -4 A>G (p > 0.05). CONCLUSIONS: The FCN2 c.772G>T genotype appears to be associated with predisposition to chronic adenotonsillitis in the pediatric age group. This nucleotide change is likely to influence the level of gene expression and contribute to the development of disease.

Integrated analysis of microRNA regulatory network in nasopharyngeal carcinoma with deep sequencing.

BACKGROUND: MicroRNAs (miRNAs) have been shown to play a critical role in the development and progression of nasopharyngeal carcinoma (NPC). Although accumulating studies have been performed on the molecular mechanisms of NPC, the miRNA regulatory networks in cancer progression remain largely unknown. Laser capture microdissection (LCM) and deep sequencing are powerful tools that can help us to detect the integrated view of miRNA-target network. METHODS: Illumina Hiseq2000 deep sequencing was used to screen differentially expressed miRNAs in laser-microdessected biopsies between 12 NPC and 8 chronic nasopharyngitis patients. The result was validated by real-time PCR on 201 NPC and 25 chronic nasopharyngitis patients. The potential candidate target genes of the miRNAs were predicted using published target prediction softwares (RNAhybrid, TargetScan, Miranda, PITA), and the overlay part was analyzed in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological process. The miRNA regulatory network analysis was performed using the Ingenuity Pathway Analysis (IPA) software. RESULTS: Eight differentially expressed miRNAs were identified between NPC and chronic nasopharyngitis patients by deep sequencing. Further qRT-PCR assays confirmed 3 down-regulated miRNAs (miR-34c-5p, miR-375 and miR-449c-5p), 4 up-regulated miRNAs (miR-205-5p, miR-92a-3p, miR-193b-3p and miR-27a-5p). Additionally, the low level of miR-34c-5p (miR-34c) was significantly correlated with advanced TNM stage. GO and KEGG enrichment analyses showed that 914 target genes were involved in cell cycle, cytokine secretion and tumor immunology, and so on. IPA revealed that cancer was the top disease associated with those dysregulated miRNAs, and the genes regulated by miR-34c were in the center of miRNA-mRNA regulatory network, including TP53, CCND1, CDK6, MET and BCL2, and the PI3K/AKT/ mTOR signaling was regarded as a significant function pathway in this network. CONCLUSION: Our study presents the current knowledge of miRNA regulatory network in NPC with combination of bioinformatics analysis and literature research. The hypothesis of miR-34c regulatory pathway may be beneficial in guiding further studies on the molecular mechanism of NPC tumorigenesis.

Epstein-Barr virus mir-bart1-5p detection via nasopharyngeal brush sampling is effective for diagnosing nasopharyngeal carcinoma.

Epstein-Barr virus (EBV)-encoded microRNAs (miRNAs) are highly expressed in nasopharyngeal carcinoma (NPC) cases in high-risk areas, and may be involved in tumorigenesis. Using quantitative RT-PCR, we detected four EBV-encoded BamHI A rightward transcript (BART) miRNAs (mir-bart1-5p, mir-bart5, mir-bart6-5p and mir-bart17-5p) exclusively in 53 NPC biopsies as compared to 69 controls. In a larger patient group, that included 215 NPC cases and 209 controls, significantly higher levels of all four EBV miRNAs were detected in tumor cells harvested directly from the nasopharynx using a less invasive nasopharyngeal (NP) brush than in the controls (p < 0.001). One EBV miRNA, mir-bart1-5p, holds particular promise for use as a diagnostic indicator of NPC (with 93.5% sensitivity and 100% specificity), and its relative expression level was reflective of disease progression. Detection of this miRNA was effective for diagnosing early-stage NPC, even in cases that were falsely diagnosed as negative based on histopathological analysis, plasma EBV DNA load, and VCA-IgA and EA-IgA titers. EBV-encoded mir-bart1-5p detection via NP brush sampling could act as an efficient and less invasive method assisting clinical diagnosis of NPC.

Expression and clinical significance of LRRC4 in benign and malignant nasopharyngeal diseases.

The aim of this study was to investigate the expression of LRRC4 in nasopharyngeal carcinomas, nasopharyngeal precancerous lesions, and nasopharyngitis as well as the clinical significance of LRRC4. Fifty patients with nasopharyngeal carcinoma were selected as study subjects; 28 patients with chronic nasopharyngitis and 22 patients with nasopharyngeal precancerous lesions served as controls. Immunohistochemical analysis was used to study protein expression of LRRC4; the relation between LRRC4 expression and the clinical stage and histopathological features of nasopharyngeal carcinoma was also analyzed. The LRRC4 expression manifested itself as yellow staining in the cytoplasm or nucleus. LRRC4 was strongly expressed in nasopharyngeal epithelial tissues of patients with chronic nasopharyngitis and in nasopharyngeal precancerous lesions; the rates of positive results were 82.1 and 81.8%, respectively. LRRC4 was weakly expressed in nasopharyngeal carcinoma tissues, at a rate of 10% positive results (P< 0.001); there was no significant difference in the expression of LRRC4 among different clinical stages and pathological grades. Therefore, disappearance of LRRC4 expression is a major feature of nasopharyngeal carcinoma.

Promoter hypermethylation along with LOH, but not mutation, contributes to inactivation of DLC-1 in nasopharyngeal carcinoma.

Previous studies have shown that promoter hypermethylation plays a key role in DLC-1 inactivation in nasopharyngeal carcinoma (NPC). However, DLC-1 mutation in NPC has not been reported, and there remain some discrepancies in methods and results between different groups. Here, we examined the mRNA and protein expression of DLC-1 in chronic nasopharyngitis (CN) and NPC tissues by reverse transcription-polymerase chain reaction/qPCR and immunohistochemistry, respectively. DLC-1 mRNA was undetectable in all the seven widely used NPC cell lines and absent or significantly down-regulated in 70% of NPC tissues. DLC-1 protein level was reduced in 74.3% of NPCs when compared with CN tissues, and significantly lower in NPC samples at advanced clinical stages than that at early stages. Then, we purified the same batch of specimens by microdissection and analyzed the possible mechanisms of DLC-1 downregulation with mutation and allelic loss analysis, methylation-specific PCR and bisulfite genomic sequencing. Only one mutation was detected at codon 693 of exon 8 in 3.3% of NPCs and five single nucleotide polymorphisms (SNPs) were identified. Loss of DLC-1 was detected in 23.3% of NPC tissues. The 100% of NPC cell lines, 80% of primary NPC and 22.2% of CN tissues showed methylation in DLC-1 promoter, while DLC-1 expression was recovered in seven NPC cell lines after 5-aza-dC treatment. Patched methylation assay confirmed that promoter methylation could repress DLC-1 expression. This report demonstrates that DLC-1 is negatively associated with NPC carcinogenesis, and promoter hypermethylation along with loss of heterozygosity, but not mutation, contributes to inactivation of DLC-1 in NPC.

Aberrant expression levels of MTA1 and RECK in nasopharyngeal carcinoma: association with metastasis, recurrence, and prognosis.

OBJECTIVES: We investigated the expression and clinical value of MTA1 and RECK genes in patients with nasopharyngeal carcinoma (NPC). METHODS: We examined MTA1 and RECK expression in nasopharyngeal tissue from patients with chronic nasopharyngitis, lymph nodes with metastasis of NPC, and primary NPC tumor tissue by means of in situ hybridization and analyzed their correlation with the clinicopathologic features of NPC. RESULTS: The positive expression of MTA1 in the NPC tissues and metastatic lymph nodes was significantly higher than that in the chronic nasopharyngitis tissues (p < 0.05). The positive expression of RECK in the NPC tissues and metastatic lymph nodes was significantly lower than that in the chronic nasopharyngitis tissues (p < 0.05). The RECK expression level was inversely correlated with the MTA1 expression level in the NPC tissues (p < 0.05). The increased MTA1 and decreased RECK expressions in the NPC tissues had no association with gender, age, T-stage, or clinical stage (p > 0.05). However, they had a positive correlation with cervical lymph node metastasis, tumor recurrence, and 5-year overall survival rate of the patients with NPC (p < 0.05). Moreover, multivariate analysis showed that MTA1 and RECK expressions were independent prognostic factors for survival (p < 0.05). CONCLUSIONS: The conversely abnormal expression levels of MTA1 and RECK may be collectively involved in progression of malignancies and may serve as molecular predictors for metastasis, recurrence, and prognosis of NPC.

Expression of miRNA-146a in nasopharyngeal carcinoma is upregulated by Epstein-Barr virus latent membrane protein 1.

We aimed to investigate the relationship between miRNA-146a and latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC). The expression levels of LMP1 in 40 cases of NPC, 28 cases of chronic nasopharyngitis and NPC cell lines CNE1 and CNE1-GL (in which LMP1 was stably transfected) were detected by immunohistochemical staining. The expression of miRNA-146a in 16 cases of NPC, 13 cases of chronic nasopharyngitis and cell lines was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. A plasmid containing the luciferase gene under the control of miRNA-146a promoter (pri-miRNA-146a) was constructed and transfected into NPC cells, and the luciferase activity was detected. LMP1 was positive in 17.9% (5/28) of chronic nasopharyngitis cases and 62.5% (25/40) of NPC cases (p<0.01). The miRNA-146a levels in NPC were significantly higher than that in chronic nasopharyngitis (p<0.01), and were higher in CNE1-GL cells than those in CNE1 cells (p<0.01). The expression of miRNA-146a in human NPC was elevated by EBV-associated antigen LMP1, probably through the activation of the miRNA-146a promoter.

Nitrative and oxidative DNA damage as potential survival biomarkers for nasopharyngeal carcinoma.

Currently, there are no satisfactory biomarkers available to screen for nasopharyngeal carcinoma (NPC). Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), has been suggested to cause nitrative and oxidative stress, leading to the accumulation of 8-nitroguanine (8-NitroG) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) and the subsequent transversion mutation of DNA. The aim of this study was to evaluate iNOS expression and the status of nitrative and oxidative stress in NPC. Fifty-nine cases of NPC and 39 cases of chronic nasopharyngitis were investigated to examine the expression of iNOS and the formation of 8-NitroG and 8-OHdG, using double-immunofluorescent staining. The statistical differences in immunoreactivities were analyzed using the Mann-Whitney test. Thirty-six patients from the 57 cases of NPC and 36 healthy controls were investigated to examine the level of serum 8-OHdG, using enzyme-linked immunosorbent assay (ELISA). The statistical differences were analyzed using a t test. Strong DNA lesions were observed in the cancer cells of NPC patients. All cases of NPC were positive for 8-NitroG and 8-OHdG, and 54 (94.7%) were positive for iNOS. NPC samples exhibited significantly more intense staining for 8-NitroG, 8-OHdG and iNOS than those of chronic nasopharyngitis (P < 0.05, respectively). The mean value of serum 8-OHdG in the 36 NPC patients was 0.538 ± 0.336 ng/ml compared to 0.069 ± 0.059 ng/ml for the healthy controls. The difference in the serum levels of 8-OHdG between the NPC patients and controls was statistically significant (P < 0.05). Our present findings suggest that pathological stimulation of nasopharyngeal tissue, caused by bacterial, viral or parasitic inflammation, may lead to nitrative and oxidative DNA lesions, caused by NO. This may contribute to the cause and development of NPC. Thus, 8-NitroG and 8-OHdG could be potential biomarkers for evaluating the risk of NPC. Better understanding of the molecular mechanisms underlying nitrative and oxidative DNA damage may provide clues to molecular targets for new approaches of NPC prevention.

[Expression, genetic and epigenetic alterations of LTF gene in nasopharyngeal carcinoma cell lines].

OBJECTIVE: To investigate the expression of LTF mRNA in several nasopharyngeal cancer (NPC) cell lines, and analyze the relationship between the genetic and epigenetic changes and expression of LTF gene. METHODS: The expression level of LTF was detected in NPC cell lines HNE1, HNE2, HNE3, CNE1, CNE2, 5-8F, 6-10B cells and tissues of 15 cases of chronic nasopharyngitis by RT-PCR. The LTF protein level was analyzed by Western blotting in 6-10B cells. Then LOH, mutation and methylation status of LTF was examined by microsatellites analysis, PCR-SSCP, MSP and bisulfite genomic sequencing, respectively. RESULTS: 15 chronic nasopharyngitis tissues showed stable LTF expression, while there were weak expression in 6-10B cells and absent expression in remaining detected NPC cell lines. There was a significantly lower LTF expression in chronic nasopharyngitis tissues (Z = -3.738, P = 0.000). No LTF protein expression was observed in 6-10B cells. LOH analysis demonstrated that allele loss of LTF wasn't found in NPC cell lines. LTF mutation was noted in 14.3% (1/7) of NPC cell lines. DNA sequencing confirmed the mutation point in the promoter region (-305 bp to -50 bp) was at -218 bp (del T) of LTF gene in the HNE1 cell line. Methylation of LTF gene was not found in chronic nasopharyngitis. However, methylation of LTF promoter was detected in all NPC cell lines. LTF mRNA expression was increased in 5-8F and 6-10B cell lines after treatment with 5-aza-2-deoxycytidine. CONCLUSION: There is an inactivation of expression of LTF gene in the NPC cell lines. Its molecular mechanism may be related with methylation of promoter region and deletion mutation.

High frequency of common deletion (4981 bp) in mitochondrial DNA in nasopharyngeal carcinoma and its correlation with patient age and clinical stages.

Mitochondrial DNA (mtDNA) has a high mutation rate due at least in part to a lack of protective histones and an inefficient DNA repair system. The most frequently change in mtDNA is the so-called Common Deletion (CD), which accumulates in patients with heteroplasmic mtDNA mutations and in normal individuals during aging. In this study, wild type mtDNA (WT-mtDNA) and mitochondrial DNA with CD (CD-mtDNA) were quantitatively analyzed in different nasopharynx lesions. A novel type of CD-mtDNA (4981 bp) was detected significantly higher in nasopharyngeal carcinoma (NPC) (93%, 54/58) than in nasopharyngitis (60%, 28/47) and the paired white blood cells (WBC) (26%, 8/31). The ratio of CD-mtDNA to WT-mtDNA in NPC (0.000625, median) was ten times that in nasopharyngitis (0.000064, median) (P=0.003), and was significantly higher than that in paired WBC (0.000000, median) (P=0.000). The CD/WT-mtDNA ratio was 0.000564 (quartile range, 0.000184-0.000919) in late stage NPC, which was nearly three times the ratio in early stage NPC (0.000164, quartile range, 0.000042-0.000353) (P=0.015, Mann-Whitney Test). In NPC patients with ages <48yrs (mean age), the ratio of CD-mtDNA to WT-mtDNA was 0.000625, which was nearly ten times that in NPC patients with ages<48yrs (0.000064) (P=0.005, Mann-Whitney Test). This is the first quantitative study of CD-mtDNA mutations in NPC, which provides evidences that CD-mtDNA mutation might be involved in the development and progression of NPC.

[Risk of meningococcal infection in children with different hla phenotypes]. / Risk zabolevaniia meningokokkovoi infektsiei detei s razlichnymi fenotipami HLA.

A total of 104 children aged 4 months to 13 years with meningococcal infection were examined. For control 600 healthy donors were used. Histocompatibility antigens were determined by the lymphotoxic test. Altogether 36 antigens of the loci, A, B and C were determined. The data thus obtained indicate that the risk of contacting the infection is 6 times greater for persons with Hl A-Bw16 phenotype and 3 times greater for persons with HL A-A12 phenotype than for persons with Hl A-A1 phenotype.