RESEARCHOpenAccessExosomalmiR-9inhibitsangiogenesisbytargetingMDKandregulatingPDK/AKTpathwayinnasopharyngealcarcinomaJuanLu1†,Qi-HuiLiu1†,FanWang1,Jia-JieTan1,Yue-QinDeng1,Xiao-HongPeng1,XiongLiu1,BaoZhang2,XiaXu3andXiang-PingLi1*AbstractBackground:Exosomesaresmallvesiclescontainingawiderangeoffunctionalproteins,mRNAandmiRNA.ExosomalmiRNAsfromcancercellsplaycrucialrolesinmediatingcell-cellcommunicationandtumor-microenvironmentcrosstalk,specificallyinenablingmetastasisandpromotingangiogenesis.WefocusedonmiR-9thatwasidentifiedasatumorsuppressorpreviouslyinnasopharyngealcarcinoma(NPC)tumorigenesis.Methods:Differentialcentrifugation,transmissionelectronmicroscopyandnanoparticletrackinganalysiswereusedtoisolateandidentifyexosomes.QuantitativePCRandwesternblottinganalysiswereusedtodetectmiR-9,pri-miR-9,CD63,TSG101,MDK,P70S6KP-Ser424andPDK1P-Ser241expression.LaserconfocalmicroscopywasusedtotraceexosomalmiR-9secretedbyNPCcellsintoHUVECs.TheeffectofexosomalmiR-9oncellmigrationandtubeformationofHUVECsinvivoandvitrowasassessedbyusingmigrationassay,tubeformationassayandmatrigelplugassay,respectively.BioinformaticsanalysisandluciferasereporterassaywereutilizedtoconfirmthebindingofexosomalmiR-9tothe3′untranslatedregion(3′-UTR)ofMDK,whilePhosphorylationArraywasperformedtoidentifyAKTPathwayinHUVECstreatedwithexosomalmiR-9.Furthermore,Immunohistochemistry(IHC)andinsituhybridization(ISH)wasusedtodetectedmiR-9,CD31andMDKexpressioninhumanNPCtumorsamples.Results:NPCcellstransfectedwithmiR-9-overexpressinglentivirus,releasedmiR-9inexosomes.ExosomalmiR-9directlysuppresseditstargetgene-MDKinendothelialcells.MechanisticanalysesrevealedthatexosomalmiR-9fromNPCcellsinhibitedendothelialtubeformationandmigrationbytargetingMDKandregulatingPDK/AKTsignalingpathway.Additionally,thelevelofMDKwasupregulatedinNPCtumorsamplesandwaspositivelycorrelatedwithmicrovesseldensity.Notably,thelevelofexosomalmiR-9waspositivelycorrelatedwithoverallsurvival,andMDKoverexpressionwaspositivelyassociatedwithpoo...
