| Nasopharyngeal carcinoma(NPC)is one of the most common cancers in southeast China.With the improvement of radiotherapy technology,the 5-year survival rate of NPC at stage I and stage II(UICC 7th edition)is 100%and 95%,respectively.The 5-year survival rate of advanced NPC was significantly lower than that of early NPC[1].There are two fundamental causes of the lower 5-year survival rate of advanced NPC.Firstly,about 60%-70%of patients were in advanced stage when they were diagnosed as nasopharyngeal carcinoma[2];secondly,local recurrence and distant metastasis severely deteriorated the outcome of patients with NPC.Epstein-Barr virus(EBV)is one of the major pathogeny of NPC.The experts’consensus of comprehensive treatment for head and neck squamous cell carcinoma in China(2013 edition)recommended that serum EBV-DNA load be used as a molecular biomarker for therapeutic monitoring,recurrence and metastasis of patients with NPC.However,the sensitivity of serum EBV-DNA was low,and some scholars pointed out that the sensitivity of EBV-DNA in peripheral blood mononuclear cells(PBMCs)was higher and could be applied to predict recurrence and metastasis of NPC[3].Therefore,it has theoretical and clinical significance to investigate the serum and PBMC EBV-DNA load in the recurrence and metastasis of NPC.Exosomes are small vesicles derived from intracellular vesicles and can carry a variety of biological molecules such as proteins,microRNAs,and mRNAs for intercellular communication and are closely related to the recurrence and metastasis of cancer.It had been reported that EBV-miR-BART7 was highly expressed in serum of patients with NPC[4].We hypothesized that EBV-miR-BART7 may secrete extracellularly to regulate cell growth through the exosome pathway.Exosomes from the serum of patients with NPC may carry EBV-miR-BART7 to participate in this process.Objectives:To investigate the role of EBV-DNA load in serum and PMBC in predicting local recurrence and/or distant metastasis of NPC,to estimate EBV-miR-BART7 as an exosome markers for NPC.Methods:Serum and whole blood samples were collected from 197 patients diagnosed with NPC by pathology and 130 healthy persons without EBV-related diseases.Among them,43 patients with local recurrence and/or distant metastasis were treated as recurrence and metastasis group.There were 154 patients who had no local recurrence and/or distant metastasis(without recurrence metastasis group).130 patients without EBV-related disease were examined as a healthy control group.The PBMCs were extracted from the whole blood samples by lymphocyte separation and the EBV-DNA load in serum and PBMCs were detected by real-time fluorescent quantitative PCR.The Kruskal-Wallis test analyzed the differences of EBV-DNA load in serum and PBMC and ratio(Q value = serum/PBMC EBV-DNA load)among the three groups.The supernatants of C666-1 cell line and NP69 cell line were ultra centrifuged to obtain exosomes.The EBV-miR-BART7 load in the exosomes was measured.The exosomes of C666-1 cell line were added to the NP69 cells to observe cell growth.The NP69 cell line exosomes were added to NP69 cells as a control.The serum of 15 patients with NPC were collected.Nine patients with NPC were recurrence and metastasis after treatment.Six patients with NPC had no recurrence and metastasis after treatment.The serum EBV-DNA load was measured and the serum was ultra centrifuged.Exosomes were obtained and the EBV-miR-BART7 load in exosomes was measured.Pearson correlation coefficient was used to analyze the correlation between serum EBV-DNA load and EBV-miR-BART7 load in exosomes.According to the level of serum exosomes EBV-miR-BART7,Q value and the EBV-DNA load,NPC patients were divided into groups and statistical analyzed the survival differences.Results:The median serum EBV-DNA load was 507.5 IU/ml in the recurrent metastasis group,which was significantly higher than 0 IU/ml(P=0.00)in the relapse-free metastasis group and 0 IU/ml in the healthy control group(p=0.00).The median EBV-DNA load in the recurrent and metastatic group was 496IU/ml,which was significantly higher than that in the non-recurrent metastasis group(138IU/ml)(P=0.01),and it was not significantly different from the healthy control group(626.5IU/ml,P=0.77).The median Q of the NPC recurrence and metastasis group was 0.87,which was higher than that of the non-recurrent metastasis group(p=0.00)and the healthy control group 0(P=0.00).The expression of EBV-miR-BART7 was detected in the supernatant of C666-1 cell line and NP69 cell line supernatant by ultracentrifugation,and the exosomes produced by the two cell lines were observed to have promotes growth effect on NP69 cells.The expression of EBV-miR-BART7 was confirmed in the supernatant of C666-1 cell line and NP69 cell line by ultra centrifugation,and the exosomes produced by both cell lines were observed to promote the rate of growth.The expression of EBV-miR-BART7 was detected in serum of 15 patients with NPC,and there was no significant correlation with serum EBV-DNA load.The survival rate of NPC patients with high serum EBV-DNA load was lower than that with low serum EBV-DNA load.The survival rate of NPC patients with high serum EBV-DNA load was lower than that with low serum EBV-DNA load,the survival rate of patients with high Q of NPC was lower than those with low Q value,while no significant difference was observed in the level of EBV-miR-BART7 load in serum exosomes.Conclusion:The level of EBV-DNA load in serum is associated with local recurrence and/or distant metastasis of NPC.The ratio of EBV-DNA load(Q value)in serum and PBMC can be used as a new biomarker for predicting recurrence or metastasis of NPC.The C666-1 cell line and the NP69 cell line exosomes have the effect of promoting cell growth.EBV-miR-BART7 can be detected in serum exosomes of patients with NPC,and EBV-miR-BART7 can be used as a marker for exosomes derived from serum of NPC. |
PDF Full Text Request