分子生物學(xué)教育課件

LRRC4,aPutativeTumorSuppressorGene,RequiresaFunctionalLeucine-richRepeatCassetteDomaintoInhibitProliferationofGliomaCellsInVitrobyModulatingthe

ExtracellularSignal-regulatedKinase/ProteinKinaseB/NuclearFactor-κBPathwayMolecularBiologyoftheCellVol.17,3534–3542,August2006GuiyuanLietal.

LRRC4genewhichcontainsaconservedleucine-richrepeat(LRR)cassetteandanimmunoglobulin(Ig)IgC2domain,isassociatedwithgliomasuppressionbothinvitroandinvivo.ThepresentstudyprovidesevidencethattheconspicuousabsenceofLRRC4inhigh-gradegliomasdirectlycontributestotheincreasingtumorgrade.ThelossofLRRC4inU251cellsiscausedbythelossofhomozygosityatchromosome7q32-ter.ItwasalsofoundthatLRRC4requiresafunctionalLRRcassettedomaintosuppressU251cellproliferation.IntheLRRcassettedomain,thethirdLRRmotifofthecoreLRRisfoundtobeindispensableforthefunctionofLRRC4.TheinhibitoryeffectofLRRC4isaccompaniedbyadecreaseintheexpressionofpERK,pAkt,pNF-Bp65,signaltransducerandactivatoroftranscriptionprotein-3(STAT3),andmutantp53,andanincreaseintheexpressionofc-JunNH2-terminalkinase(JNK)2andp-c-Jun,suggestingthatLRRC4playsamajorroleinsuppressingU251cellproliferationbyregulatingtheextracellularsignal-regulatedkinase(ERK)/Akt/NF-Bp65,STAT3,andJNK2/c-Junpathways.TumorSamplesandCellLinesNucleicAcidIsolationandRNAAnalysisOne-StepPCRMutagenesisandConstructsWesternBlottingAnalysisCellProliferationAssaySoftAgarAssayCellCycleAnalysisAcridineOrange(AO)/EthidiumBromide(EB)StainingStatisticalAnalysisFigure1.AnalysisofLRRC4expressioningliomasandglioblastomacelllines.(A)NorthernblottinganalysisofLRRC4inglioblastomacelllinesandgliomas(top).RelativelevelsofRNAloadingareshownasmethylenebluestainingof28sRNAandGAPDH(bottom).(B)RT-PCRanalysisofLRRC4expressioningliomas,primaryculturegliomacells,andglioblastomacelllines.RNAfromgliomasandcelllineswasamplifiedfor25and35cycles,respectively,usingGAPDH(bottom)andLRRC4(top)primers.Waterwasusedasthenegativecontrol,andfetalbraincDNAandpcDNA3.1()-LRRC4plasmidwereusedasthepositivecontroltemplates.Lanes1–4,primaryculturetumorcellsderivedfromgradeII–IIIglioma;andlane5,primaryculturetumorcellsderivedfromgradeIVglioblastoma.LRRC4arrestsU251cellsinG0/G1phase,itispossiblethatLRRC4alsoinducesU251cellsapoptosis.WeusedtheanalysisofflowcytometryandAO/EBdualstainingtodetectU251cellapoptosis.AnAO/EBcocktail(80l)containing1mlofDMEMwasaddedinthecultureplate.Fieldsofstainedcellswereselectedandfocusedusingfluorescencemicroscopy(NikonEclipseE800;Nikon,Tokyo,Japan).ViablecellsstainedonlywithAOwerebrightgreenwithintactstructure;earlyapoptoticcellsstainedwithAO/EBwerebrightgreeninthenucleuswithred-orangechromatin.LateapoptoticcellsstainedwithbothAOandEBwerered-orangewithchromatincondensation.ItwasshownthatLRRC4inducednecrosis,butnotapoptosisInconclusion,LRRC4mayactasanovelcandidateoftumorsuppressorgen（TSG）.maybeinvolvedinthepathogenesisofgliomasandthet