MicroRNA-4721,Induced By EBV-miR-BART22,Targets GSK3? To Enhance The Tumorigenic Capacity Of Nasopharyngeal Carcinoma Through WNT/?-catenin Pathway

BackgroundsNasopharyngeal carcinoma(NPC)is a malignant tumor that occurs on the top and side walls of the nasopharyngeal cavity.The incidence of nasopharyngeal carcinoma is especially high in Guangdong,Guangxi,Hunan and other regions.Early-stage nasopharyngeal carcinoma patients have higher cure rates and better prognosis due to less tumor burden and higher sensitive to medical therapy;However,middle and advanced-stage nasopharyngeal carcinoma patients often fail to meet their·expected prognosis because of extensive lesions,distant metastasis and poor response to radiotherapy and chemotherapy.miR-4721 is a differentially expressed miRNA screened and discovered from EBV-miR-BART22 chip.Our previous work has proved its relation with tumorigenic capacity of nasopharyngeal carcinoma.However,the expression,molecular function and regulatory mechanism of miR-4721 have not been studied so far.Contents and methods1.EBV-miR-BART22 chip screening and miR-4721 expression detection(1)Construction of overexpressing EBV-miR-BART22 cells:Transient transfection of EBV-miR-BART22 mimic and NC mimic;(2)miRNA chip:Agilent miRNA chips labeling,hybridization and screening.Raw data was obtained for normalization and standardization.120 differentially expressed miRNAs were filtered for secondary verification and hierarchical clustering analysis;(3)Clinical sample testing:qPCR experiment was used to detect the expression of miR-4721 in 15 cases human nasopharyngeal epithelial tissues and 20 cases nasopharyngeal carcinoma tissues.2.Explore the function of miR-4721 in nasopharyngeal carcinoma(1)MTT,EDU and colony formation experiments were conducted to explore the role of miR-4721 in the proliferation of NPC cells;(2)Cell cycle experiment was conducted to detect the function of miR-4721 in the cell cycle of NPC cells;(3)Subcutaneous tumor formation experiment was performed to explore the role of miR-4721 in the growth of NPC.3.Explore the target genes and pathways of miR-4721(1)Bioinformatic websites were used to predict the target genes of miR-4721 and its binding sequence;(2)qPCR,western blot and immunohistochemistry experiments were used to explore the regulatory effect of miR-4721 on GSK3?;(3)The dual luciferase reporter gene experiment was conducted to confirm combination between miR-4721 and GSK3?;(4)Immunofluorescence detection of miR-4721's effect on ?-catenin nucleus transportation;(5)Western blot and functional experiments were conducted to verify the activation of miR-4721 on WNT/?-catenin pathway.4.Explore the upstream regulatory signal of miR-4721(1)Bioinformatics websites were used to predict the transcription factor and its binding site;(2)qPCR experiment was conducted to explore the regulatory effect of transcription factor SP1 on miR-4721;(3)Chromatin immunoprecipitation(ChIP),EMSA and dual luciferase reporter gene experiments were performed to detect the transcriptional binding between SP1 and miR-4721;(4)Co-immunoprecipitation(CoIP)and Chromatin immunoprecipitation(ChIP)experiments were performed to verify the effect of C-JUN on SP1-miR-4721 regulation;(5)qPCR experiment was used to explore the regulation of EBV-miR-BART22 and C-JUN on miR-4721.5.Analyze the expression characteristics and correlation of miR-4721 and GSK3? in nasopharyngeal carcinoma tissues(1)In situ hybridization and immunohistochemistry experiments were conducted explore the clinical characterization of miR-4721 and GSK3?;(2)Analysis of the expression correlation between miR-4721 and GSK3?.Results1.miR-4721 is highly expressed in nasopharyngeal carcinoma and induced by EBV-miR-BART22;2.miR-4721 promotes the proliferation and cell cycle transition of nasopharyngeal carcinoma in vivo and in vitro;3.miR-4721 targets GSK3? to activate WNT/?-catenin pathway;4.miR-4721 was induced by EBV-miR-BART22/C-JUN/SP1 signal axis;5.miR-4721 is negatively correlated with the expression of GSK3?;NPC patients with lower miR-4721 and higher GSK3? expression have better prognosis.ConclusionsmiR-4721 is highly expressed in nasopharyngeal carcinoma,is induced by EBV-miR-BART22/C-JUN/SP1 signal axis to target GSK3? and activate the WNT/?-catenin pathway and promote the malignant progression of nasopharyngeal carcinoma.