| Purpose:Nasopharyngeal carcinoma(NPC)is prevalent in Southeast Asia and Southern China,and radiotherapy is considered as the primary treatment for NPC.Currently,the 5-year overall survival(OS)rate for early stage NPC can exceed 95%.However,OS rates for locally advanced NPC were merely 78.4~80%,and 20~30% of which still develop local failure and distant metastasis.Therefore,it is important to investigate the pathogenesis and explore the potential serum biomarkers in NPC for early diagnosis.Increasing evidence has demonstrated that dysregulation of microRNAs(miRNAs)is the hallmark of cancer.NPC is thought to be the ubiquitous correlation with Epstein-Barr virus(EBV).Few EBV encoded virus proteins could be expressed in NPC epithelial cells,but EBV encoded BARTs miRNAs were found to be expressed abundantly in NPC cells.Our previous study showed that miR-BART13 was expressed at high levels in C666-1 cell,and it could be secreted into the extracellular environment as well.Furthermore,the level of miR-BART13 was distinctly higher than non-NPC controls,and highly various levels of miR-BART13 were positively correlated with advanced N stage and clinical stage for NPC.Hence,miR-BART13 may be a potential serum biomarker for early diagnosis,therapeutic evaluation,and prognostic assessment of NPC.It is necessary to have a further investigation of the molecular mechanisms of miR-BART13 in NPC initiation and progression,so as to provide theory basis for miR-BART13 to be a potential biomarker and therapeutic target in clinical practice.Therefore,the aim of our present study was mainly to investigate the mechanisms of miR-BART13 on immune evasion,tumor invasion and metastasis in nasopharyngeal carcinoma in vitro.Materials and methods:1.After stable overexpression of miR-BART13 in CNE-1 without EBV infection by LV-miR-BART13 or stable downregulation of miR-BART13 in C666-1 which harbored EBV by LV-miR-BART13 sponge,those target NPC cells were thenco-cultured with NK cells for 4 hours,respectively.Flow cytometry and LDH were taken to reflect the cytotoxcity of NK cells against NPC cells.In addition,ELISA and flow cytometry were employed to reflect the expression levels of IFN-? and CD107 a produced by NK cells induced by target NPC cells.2.To investigate the regulating function of miR-BART13 on ULBP4,luciferase reporter assays,RT-qPCR,Western Blot,and flow cytometry were employed.ULBP4 expression in NPC and NP tissues at mRNA and protein levels was tested by RT-qPCR and immunohistochemistry(IHC),and the correlations between different expression levels of ULBP4 protein and clinical characteristics in 111 patients with NPC were evaluated,so were those of ULBP4 protein and disease prognoses.After5-8F and C666-1 cells were infected with LV-ULBP4,LDH was used to reflect the impact of the cytotoxic activity of NK cells on target cells.In addition,After partially rescuing ULBP4 expression on CNE-1-BART13 or C666-1-BART13 sponge cells,flow cytometry was used to detect the cytotoxic activity of NK cells on NPC cells,and ELISA and flow cytometry were employed to reflect the ability of NK cells producing IFN-? and CD107 a.3.After stable overexpression of miR-BART13 in CNE-1 or stable downregulation of miR-BART13 in C666-1,the alternative ability of migration and invasion in NPC cells were analyzed by the wound healing assay,transwell migration,and transwell invasion.Morphologic changes of NPC cells were also observed.Furthermore,the alternative expression levels of protein markers on tumor metastases and EMT were tested by Western Blot.4.With a combination of bioinformatics analysis and cell ITRAQ,we tried to find miR-BART13' direct targets which were shown to suppress the tumor metastases.Based on these findings,these possible direct targets were further validated by RT-qPCR and Western Blot.In addition,Western Blot was used to analyze the impact of miR-BART13 on the signaling pathway proteins associated with the possibly direct targets.Results:1.After CNE-1 stably overexpressed miR-BART13,flow cytometry showed thecytotoxicity of NK cells was significantly increased,the expression of IFN-? and CD107 a were also considerably increased at the effector/target(E/T)ratio of 10:1.In addition,the cytotoxic sensitivity of CNE-1-BART13 cells against NK cells was higher than that of CNE-1-vector cells at the three E/T ratios(10:1,20:1,40:1).However,after C666-1 stably downregulated miR-BART13,the cytotoxic activity of NK cells were contrary to the above results by the same methods.2.ULBP4 was found to be a direct target of miR-BART13 by luciferase reporter assays,Western Blot,RT-qPCR,and flow cytometry.ULBP4 was significantly decreased in primary NPC tissues as compared to NP tissues at both mRNA and protein levels,and low expression of ULBP4 in NPC was demonstrated to be of potentially independent significance for predicting OS,DFS,and DMFS.LDH analysis showed that the cytotoxicity of NK cells against NPC cells(5-8F or C666-1)with LV-ULPB4 was significantly increased when compared with those with LV-vector at the E/T ratios of 10:1,20:1,40:1,respectively.To have further “rescue”experiments,after CNE-1-BART13 cells overexpressed ULBP4,flow cytometry showed the cytotoxicity of NK cells was significantly increased,the expression of IFN-? and CD107 a were considerably increased as well at E/T ratio of 10:1.However,after C666-1 stably downregulated miR-BART13,the cytotoxic activity of NK cells were opposite to the above results with the same methods.3.After CNE-1 stably overexpressed miR-BART13,the migration and invasion abilities of CNE-1 cells considerably improved,MMP9 protein was upregulated,and TIMP2 protein was downregulated;morphology of CNE-1 cells presented to be kinds of slender;EMT markers like E-cadherin protein was downregulated,whereas Vimentin?N-cadherin?ZEB1??-catenin proteins were upregulated.However,after C666-1 stably downregulated miR-BART13,the results were contrary to the above findings,and morphology of C666-1 cells presented to be kinds of elliptic.4.By cross-over analysis of bioinformatics and ITRAQ,NIKRAS2 might be a potential target of miR-BART13.RT-qPCR and Western Blot showed that NKIRAS2 mRNA and protein levels were upregulated in C666-1 cells after miR-BART13 being downregulated.To have a deep research about the influence of miR-BART13 onNF-?B signaling pathway,after miR-BART13 of C666-1 was downregulated,I?B?and I?B? proteins were increased,while p-NF-?B(p65)and nuclear NF-?B(p65)proteins were reduced.Nevertheless,after miR-BART13 of CNE-1 was upregulated,the results were contrary to the above findings.Conclusions:1.miR-BART13 decreases the cytotoxicity of NK cells to promote tumor immune evasion by suppressing the NKG2 D ligand ULBP4.2.ULBP4 was significantly reduced in primary NPC tissues at both mRNA and protein levels,and low expression of ULBP4 was of potentially independent significance for predicting OS,DFS,and DMFS.3.miR-BART13 promotes the EMT and metastasis of NPC cells by activating NF-?B signaling pathway. |
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