Preliminary Study On The Mechanism Of Epstein-barr Virus Encoded MiR-BART7-3p On Immune Evasion In Nasopharyngeal Carcinoma

Background and objective: Nasopharyngeal carcinoma(NPC)occurs predominantly in southern China and Southeast Asian countries.For early and locally advanced NPC,radiotherapy is the main treatment method.In recent years,with the development of radiotherapy technology,and the combined use of chemotherapy and targeted therapy,the therapeutic outcomes of NPC have been remarkably improved.However,about 20% of the NPC patients develop treatment failure due to local recurrence and distant metastasis.Many studies have shown that EBV infection was closely associated with the development of NPC.EBV can encode 44 mature BART mi RNAs in NPC.Of them,the expression of EBV-mi R-BART7-3p was the most abundant,which indicated that it may play an important role in tumor initiation and progression.Actually,several studies have revealed EBV-mi R-BART7-3p overexpression can promote the proliferation,invasion and metastasis ability of NPC cells.Cancer cells can escape the immune recognition and elimination by the host immune system through multiple mechanisms,and may further promote the invasion and metastasis of tumor cells,which may be the cause of treatment resistance and recurrence.There have been no reports of the relationship between EBV-mi R-BART7-3p and immune evasion in NPC to date.In the present study,we aimed to preliminarily investigate the role of EBV-mi R-BART7-3p played in immune evasion in NPC,and to find a new target for the diagnosis and treatment of NPC.Methods: We firstly compared the differential expression of EBV-mi R-BART7-3p between nasopharyngeal carcinoma and normal nasopharyngeal mucosa tissues by real-time PCR.And then we constructed CNE1 cell line with stable overexpression of EBV-mi R-BART7-3p via lentivirus vectors.Peripheral blood mononuclear cells were obtained from healthy donors.NK cells were cultured in the combination of IL-2+IL-12+IL-15+IL-18.And then NK cells were sortedand purified by Vario MACS.We compared the cytotoxicity of NK cells against CNE1-BART7 and CNE1-vector by LDH assay,detecting the ralease of IFNγ by ELISA and the expression of CD107 a in NK cells by FACS.We conjunctively used bioinformatics sofeware Target Scan and mi Randa to predict and identify the potiential targets of EBV-mi R-BART7-3p.Then luciferase reporter assay was performed to validiate the direct regulator gene sites.Furthermore,the m RNA and protein levels of ULBP4 in EBV-mi R-BART7-3p overexpressed human nasopharyngeal carcinoma cell line CNE1 or tissues were detected by real-time PCR,western blot and antibody-labeled flow cytometry.The expression of ULBP4 in four nasopharyngeal carcinoma cell lines were also detected by real-time PCR and Western blot.So as to confirm the regulating effect of EBV-mi R-BART7-3p on the target gene ULBP4.Finally,we used rescue assay to further verify EBV-mi R-BART7-3p participate in nasopharyngeal carcinoma immune evasion by down-regulatiing the expression of ULBP4.Results: Real-time PCR results displayed that the expression of EBV-mi R-BART7-3p in NPC tissues was higher than that in NP tissues.Cytotoxic effects of NK cells against CNE1-BART7 cells were significantly higher than that against CNE1-vector cells.Afterwards,we identified a potential EBV-mi R-BART7-3p binding site in the 3’UTR of human ULBP4 gene through bioinformatics analysis.The luciferase reporter assay showed that EBV-mi R-BART7-3p could bind directly to the specific site of ULBP4 m RNA 3’UTR.Overexpression EBV-mi R-BART7-3p into CNE1 cells significantly reduced the ULBP4 m RNA and protein levels.The expression of ULBP4 in NPC tissues was higher than that in NP tissues,and the expression of ULBP4 in EBV-negative nasopharyngeal carcinoma cell lines was higher than that in EBV positive nasopharyngeal carcinoma cell lines.Upregulation of ULBP4 in CNE1-BART7 cells could partially recuse the cytotoxicity activity of NK cells.Recuse experiments,on the other hand,confirmed that ULBP4 was indeed regulated by EBV-mi R-BART7-3p.Conclusion: Our study demonstrated that EBV-mi R-BART7-3p was significantly higher in NPC tissues than in NP tissues.The susceptibility of CNE1 cells overexpression with EBV-mi R-BART7-3p genes to NK cell-mediated cytotoxicity were decreased significantly.EBV-mi R-BART7-3p can be directly bound to the EBV-mi R-BART7-3p binding sites in the 3’UTR of ULBP4 m RNA,and inversely correlated with the expression of ULBP4 in NPC.NPC cells could down-regulate the expression of ULBP4 by up-regulating EBV-mi R-BART7-3p,and thus escape the immune surveillance,which may promote the development of NPC.And this pathway may provide a potential new target for the diagnosis and treatment of NPC.