Exosomes are small vesicles containing a wide range of functional proteins, mRNA and miRNA. Exosomal miRNAs from cancer cells play crucial roles in mediating cell-cell communication and tumor-microenvironment cross talk, specifically in enabling metastasis and promoting angiogenesis. We focused on miR-9 that was identified as a tumor suppressor previously in nasopharyngeal carcinoma (NPC) tumorigenesis. Differential centrifugation, transmission electron microscopy and nanoparticle tracking analysis were used to isolate and identify exosomes. Quantitative PCR and western blotting analysis were used to detect miR-9, pri-miR-9, CD63, TSG101, MDK, P70S6K P-Ser424 and PDK1 P-Ser241 expression. Laser confocal microscopy was used to trace exosomal miR-9 secreted by NPC cells into HUVECs. The effect of exosomal miR-9 on cell migration and tube formation of HUVECs in vivo and vitro was assessed by using migration assay, tube formation assay and matrigel plug assay, respectively. Bioinformatics analysis and luciferase reporter assay were utilized to confirm the binding of exosomal miR-9 to the 3′untranslated region (3′-UTR) of MDK, while Phosphorylation Array was performed to identify AKT Pathway in HUVECs treated with exosomal miR-9. Furthermore, Immunohistochemistry (IHC) and in situ hybridization (ISH) was used to detected miR-9, CD31 and MDK expression in human NPC tumor samples. NPC cells transfected with miR-9-overexpressing lentivirus, released miR-9 in exosomes. Exosomal miR-9 directly suppressed its target gene - MDK in endothelial cells. Mechanistic analyses revealed that exosomal miR-9 from NPC cells inhibited endothelial tube formation and migration by targeting MDK and regulating PDK/AKT signaling pathway. Additionally, the level of MDK was upregulated in NPC tumor samples and was positively correlated with microvessel density. Notably, the level of exosomal miR-9 was positively correlated with overall survival, and MDK overexpression was positively associated with poor prognosis in NPC patients, suggesting the clinical relevance and prognostic value of exosomal miR-9 and MDK. Taken together, our data identify an extracellular anti-angiogenic role for tumor-derived, exosome-associated miR-9 in NPC tumorigenesis and prompt further investigation into exosome-based therapies for cancer treatment.

The molecular mechanisms underlying dysregulation of microRNAs have been documented in nasopharyngeal carcinoma (NPC). Our previous study demonstrated that plasma miR-124 was down-regulated in NPC using microarray analysis and quantitative PCR validation. Though growing studies showed that down-regulated miR-124 was closely related to tumourigenesis in various types of cancers, the role of miR-124 in NPC remains largely unknown. The expression level of miR-124 was evaluated in NPC cell lines and patient specimens using quantitative reverse transcription-PCR (Real-time qPCR). The clinicopathological significance of the resultant data was later analyzed. Then, we explored the role of miR-124 in NPC tumorigenesis by in vitro and in vivo experiments. Homo sapiens forkhead box Q1 (Foxq1) was confirmed as a novel direct target gene of miR-124 by the dual-luciferase assay and western bolt. We found that miR-124 was commonly down-regulated in NPC specimens and NPC cell lines. The expression of miR-124 was inversely correlation with clinical stages and marked on T stages. Then, the ectopic expression of miR-124 dramatically inhibited cell proliferation, colony formation, migration and invasion in vitro, as well as tumor growth and metastasis in vivo. Furthermore, we identified Foxq1 as a novel direct target of miR-124. Functional studies showed that knockdown of Foxq1 inhibited cell growth, migration and invasion, whereas Foxq1 overexpression partially rescued the suppressive effect of miR-124 in NPC. In clinical specimens, Foxq1 was commonly up-regulated in NPC, and the level increased with clinical stages and T stages. Additionally, the level of Foxq1 was inversely correlated with miR-124. Our results demonstrate that miR-124 functions as a tumor-suppressive microRNA in NPC, and that its suppressive effects are mediated chiefly by repressing Foxq1 expression. MiR-124 could serve as an independent biomarker to identify patients with different clinical characteristics. Therefore, our findings provide valuable clues toward the understanding the of mechanisms of NPC pathogenesis and provide an opportunity to develop new effective clinical therapies in the future.

Background Nasopharyngeal carcinoma (NPC) is primarily caused by the Epstein-Barr virus (EBV) infection in NPC endemic areas. EBNA1 is an EBV-encoded nuclear antigen, which plays a critical role in the maintenance and replication of EBV genome. However, the mechanisms of EBNA1-promoted NPC immune escape are unknown. Regulatory T (Treg) cells are among the key regulators in restraining antitumor responses. However, the mechanisms of accumulation of Treg cells in NPC have not been defined. This study attempted to identify the detailed mechanisms of EBNA1 functions as a tumor accelerator to promote NPC immune escape by enhancing chemoattraction of Treg cells. Methods mRNA profiles were determined by next-generation sequencing in NPC cells. In vitro and in vivo assays were performed to analyze the role of EBNA1 in regulation of recruitment of Treg cells. Colocation and coimmunoprecipitation analyzes were used to identify the SMAD3/c-JUN complex. Chromatin immunoprecipitation assay and dual luciferase reporter assays were designed to demonstrate c-JUN binding to miR-200a promoter and miR-200a targeting to CXCL12 3’Untranslated Regions. The hepatocellular carcinoma models were designed to demonstrate universality of the CXCL12-CXCR4-Treg axis in promoting immune evasion of various tumors. Result A novel molecular mechanism was identified that involves EBV-EBNA1-stimulated chemotactic migration of Treg cells toward NPC microenvironment by upregulation of the transforming growth factor-β1 (TGFβ1)-SMAD3-PI3K-AKT-c-JUN-CXCL12-CXCR4 axis and downregulation of miR-200a. EBV-EBNA1 promotes the chemoattraction of Treg cells by governing the protein–protein interactions of the SMAD3/c-JUN complex in a TGFβ1-dependent manner in vitro and in vivo. TGFβ1 suppresses miR-200a by enhancing the SMAD3/c-JUN complex. miR-200a negatively regulates the CXCL12 chemokine by direct targeting of the CXCL12 3’UTR region. However, CXCL12 acts as the target gene of miR-200a and as an inhibitor of miR-200a transcription, and inhibition of miR-200a by CXCL12 is mediated by c-JUN, which directly binds to the miR-200a promoter and forms a c-JUN-miR-200a-CXCL12-c-JUN feedback loop. In addition, enhanced CXCL12 efficiently attracts CXCR4-positive Treg cells to remodel an immunosuppressive microenvironment. Conclusions EBV-EBNA1 promotes chemotactic migration of Treg cells via the TGFβ1-SMAD3-PI3K-AKT-c-JUN-miR-200a-CXCL12-CXCR4 axis in the NPC microenvironment. These results suggest that EBV-EBNA1 may serve as a potential therapeutic target to reshape the NPC microenvironment.

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.

Nasopharyngeal carcinoma (NPC) is uncommon worldwide but often highly invasive in late stages. Due to its special location and lack of specific symptoms, NPC is hardly detected in regular medical examination at the beginning. Development of sensitive and specific biomarkers should help to save lives against this type of disease. In the present report, we investigated the value of plasma miRNAs for diagnosis and prognosis of NPC. Using candidate approach, we selected 21 miRNAs from literature to compare their expression levels in the plasma of NPC patients and controls. As a result, 5 miRNAs showed diagnostic potentials (P < 0.01). Among them, miR-16, -21, -24, and -155 had increased levels in NPC patients, whereas the level of miR-378 was decreased. There was a negative correlation between plasma miRNA expression and cancer progression, where miR-21 was statistically significant in T and N staging and miR-16 and 24 were significant in N staging only. Combination of miR-16, -21, -24, -155, and -378 gives 87.7% of sensitivity and 82.0% of specificity for NPC diagnosis. Without miR-16, combination of the rest 4 miRNAs gives the same sensitivity but a slightly reduced specificity. After treatment, all 5 miRNAs were somewhat back to normal levels in patients without cancer recurrence but the prognostic value was not statistically significant. In conclusion, plasma miRNA expression is a useful biomarker for NPC diagnosis but not for its prognosis. More importantly, it is simple, effective, and non-invasive. Combination of several plasma miRNAs can increase both NPC diagnostic sensitivity and specificity.

Background: This study was performed to investigate whether long‐term monitoring of dynamic changes in plasma Epstein‐Barr virus (EBV) DNA could improve prognosis prediction of nasopharyngeal carcinoma (NPC).Materials and methods: About 1077 nonmetastatic NPC patients were recruited to retrospectively analyze the prognostic value of plasma EBV DNA load pretreatment and 3, 12, 24, and 36 months posttreatment. We also examined the prognostic value of dynamic changes in plasma EBV DNA at various time points.Results: Patients with plasma EBV DNA load above optimal pre‐ and posttreatment cut‐offs had significantly worse five‐year progression‐free survival, distant metastasis‐free survival, locoregional relapse‐free survival, and overall survival (OS) at all‐time points, excluding only OS at 36 months posttreatment due to limited mortalities. Patients with persistently undetectable plasma EBV DNA at the first four time points had the best prognosis, followed by those with positive detection pretreatment and consistently negative detection posttreatment, those with negative detection pretreatment and positive detection at one time point posttreatment, and those with positive detection pretreatment and at one time point posttreatment, whereas patients with positive detection at ≥2 time points posttreatment had the worst prognosis. Cox proportional hazard models identified the dynamic change pattern as an independent prognostic factor, and receiver operating characteristic curve analysis demonstrated that the dynamic change at four time point was more valuable than any single time point for predicting disease progression, distant metastasis, locoregional relapse, and mortality.Conclusions: Dynamic changes in plasma EBV DNA pre‐ and posttreatment could predict the long‐term survival outcome and provide accurate risk stratification in NPC.