| Background and ObjectivesNasopharyngeal carcinoma is an epithelial carcinoma arising from the nasopharyngeal mucosal lining.Nasopharyngeal carcinoma has high degree of malignancy,and is apt to early metastasis and recurrence.The risk factors of nasopharyngel carcinoma include genetic susceptibility,environmental exposure to air dust pollution and EB virus infection.BamHI A rightward transcripts(BARTs)are particularly abundant in EB V-associated carcinomas and encode a large number of miRNAs involved in tumor progression and treatment.Therefore,the study of EB virus-related important genes or miRNAs and their signaling pathways will further reveal the pathogenesis of nasopharyngeal carcinoma.Cancer cell resistance to chemotherapeutics is the leading cause of death in cancer patients.Reduction of effective drug concentration in cells,impaired repair of DNA damage,mutations and abnormal expression of genes,and abnormalities in signal transduction pathways are all involved in multidrug resistance of tumor cells.In this study,we examined the relationship between EBV-miR-BART22 and MAP2K4 in NPC and found an atypical MYH9/GSK3?/?-catenin pathway mediated tumor stemness and EMT signal to induce cisplatin resistance.Together these results provide a new idea for the molecular mechanism of nasopharyngeal carcinoma.Contents and methods1.Using in vitro and in vivo experiments to detecte the effect of NPC by EBV-miR-BART22 and its molecular mechanism2.Bioinformatics,Luciferase reporter assay and Ago2-RIP were used to detecte the target gene of EBV-miR-BART22.3.EMSA,ChIP assay,Luciferase reporter assay and Western Blot were used to explore the mechanism of MAP2K4 reduces MYH9 expression by downregulating PI3K/AKT/c-Jun-mediated stimulation4.To study whether the MYH9 promotes GSK3? protein ubiquitin degradation,and the molecular mechanism that MYH9 activate the Wnt/?-catenin signaling.(1)Co-IP and Immunofluorescence were used to detect the interaction and localization between MYH9 and GSK3? in nasopharyngeal carcinoma cells;(2)Cycloheximide(CHX)chase assay was performed to investigate the effect of MYH9 on the the stability of GSK3? in NPC cells.(3)Co-IP was applied to observe the effect of MYH9 on ubiquitination of GSK3?;(4)Using immunofluorescence to observe the effects of over-expression or silencing of MYH9 on ?-catenin localization and detected the nuclear plasma separation of?-catenin.5.Explore the mechanism of MYH9 regulates Ubiquitin expression by PI3K/AKT/c-Jun-mediated stimulation(1)Bioinformatics software was used to predict the transcription start site of Ubiquitin;real-time PCR,EMSA,ChIP and Luciferase reporter assay were used to confirm the binding of c-Jun in the transcription regulatory region of Ubiquitin;(2)Western Blot was to detect PI3K/AKT/c-Jun/Ubiquitin protein changes in nasopharyngeal carcinoma cells overexpressing MYH9 or treated with LY294002 inhibitors;(3)ChIP assay was performed to confirm the combination of c-Jun in the transcription regulatory region of Ubiquitin after treated with LY294002 inhibitors;6.Using the functional experiments to investigate whether MAP2K4 can antagonize the biological function and molecular mechanism of EBV-miR-BART22 in nasopharyngeal carcinoma cells.(1)Tumor sphere formation,Transwell,Boyden Chamber assays,the DDP chemo-resistance assay and Western Blot were conducted to detect the stemness,invasiveness,metastasis,and DDP resistance in miR-BART22-overexpressing NPC cells after transfected with MAP2K4 plasmids;(2)Co-IP was applied to observe the effect of MAP2K4 on the interaction between GSK3? and Ubiquitin in miR-BART22-overexpressing NPC cells;(3)ChIP assay was performed to confirm the combination of c-Jun in the transcription regulatory region of Ubiquitin and MYH9 after treated with MAP2K4 in miR-BART22-overexpressing NPC cells;7.Using the functional experiments to investigate whether Knocking down MYH9 can antagonize the biological function and molecular mechanism of EBV-miR-BART22 in nasopharyngeal carcinoma cells.8.Analysis the expression and correlation of EBV-miR-BART22,MAP2K4 in nasopharyngeal carcinoma tissues,and the influence on prognosis and relationship with clinical pathological parameters.Results(1)EBV-miR-BART22 activates PI3K/AKT/c-Jun and GSK3?/?-catenin signaling pathways to promote tumor stemness,invasion and metastasis,and resistance to cisplatin in nasopharyngeal carcinoma;(2)EBV-miR-BART22 directly targets MAP2K4;(3)MAP2K4 inhibits MYH9 transcriptional regulation by down-regulating PI3K/AKT/c-Jun-mediated signaling pathways;(4)MYH9 interacts with GSK3? and promotes GSK3? protein ubiquitin degradation;(5)MAP2K4 antagonizes the action of EBV-miR-BART22 in nasopharyngeal carcinoma;(6)Knocking down MYH9 antagonizes the action of EBV-miR-BART22;(7)EBV-miR-BART22 expression was markedly upregulated in NPC tissues,and MAP2K4 was significantly lower in NPC than in NP samples;NPC patients with low MAP2K4 expression and high expression of miR-BART22 had the worst survival prognosis... |
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