Figure 1 | Oncogene

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From:Gain-of-functionmutantp53downregulatesmiR-223contributingtochemoresistanceofculturedtumorcells

Mutantp53negativelyregulatesmiR-223expression.(a)Expressionofmutantp53R175HinthelungcelllineH1299leadstomiR-223downregulation.ThecelllineH1299cloneno.41wastreatedwith2.5鈥壩?span>MponasteroneA(ponA)and10鈥壩糶/mlcisplatin(CDDP)toinducetheexpressionofandactivatemutantp53R175H,asdescribedpreviously.14,17,21AnaliquotofcellstreatedornotwithponAwerelysedtoobtainproteinextractsandrunonsodiumdodecylsulfate鈥損olyacrylamidegelelectrophoresis(SDS鈥揚AGE)toconfirmp53R175Hinduction.Arepresentativewesternblot,inwhichponceaustainingofthemembranewasusedasloadingcontrol,isshownintherightpanel.Uponinductionofp53R175H,theexpressionofmaturemiR-223substantiallydecreased.miR-223expressionlevelsweremeasuredbyreal-timepolymerasechainreaction(PCR)withtheTaqManMicroRNAassay(AppliedBiosystems,FosterCity,CA,USA)followingthemanufacturer鈥檚instructions.Analysisofthedatawasperformedbythe螖螖Ctmethod,usingRNU6B(TaqManMircroRNAassay;AppliedBiosystems)asendogenouscontrolforstandardization.(bandc)Downregulationofmutantp53indifferentcancercelllinesincreasesmiR-223expression.(b)ThecolonandbreastcancercelllinesSW480andMDA-MB-468,whichexpress,respectively,mutantp53R273H,P309Sandp53R273H,werestablytransfectedwithplasmidsexpressingshort-hairpinRNAsagainstp53(sh53)orwithascrambledcontrol(shSC).16TransfectionwasperformedusingLipofectamine2000(Invitrogen,Carlsbad,CA,USA)followingthemanufacturer鈥檚instructions.Cellswereselectedwith2鈥壩糶/mlpuromycin.(c)Mutantp53wastransientlysilencedinthesamecelllinesplusthebreastcancerlineMDA-MB-231(expressingp53R280K)bytransfectionofsiRNAagainstp53(sip53)oragainstthegreenfluorescentproteinasacontrol(siGFP)(MWG,Ebersberg,Germany;siRNAp53:5鈥?GACUCCAGUGGUAAUCUAC-3鈥?siRNAGFP:5鈥?AAGUUCAGCGUGUCCGGGGAG-3鈥?.Downregulationofmutantp53wascheckedbywesternblotwithananti-p53antibody(mousemonoclonalDO1)usingactin(anti-actinAC74;Sigma-Aldrich,StLouis,MO,USA)orglyceraldehyde3-phosphatedehydrogenase(GAPDH)(anti-GAPDH;SantaCruzBiotechnology,Dallas,TX,USA;SC32233)forstandardization;secondaryantibodies,horseradishperoxidase(HRP)-conjugatedanti-rabbitandanti-mouse,wereobtainedbyBio-RadLaboratories(Hercules,CA,USA).DetectionofthewesternblotswasperformedwiththeImmobilonWesternreagent(Millipore,Hayward,CA,USA)andimagesoftheblotswereobtainedwithVersaDocModel4000,usingtheQuantityOnesoftware(Bio-RadLaboratories).Representativewesternblotareshowninlowerpanels.UponRNAisolationbyTrizolreagent(Invitrogen),miR-223expressionlevelsweremeasuredbyreal-timePCRusingthemiScriptRTkitandmiScriptPCRsystem(Qiagen,Hilden,Germany)(upperpanels).Analysisofthedatawasperformedbythe螖螖Ctmethod,usingRNU6B(miScriptPCRsystem;Qiagen)asanendogenouscontrolforstandardization(n=2卤s.e.m.).Inalltheexperimentalsettings,mutantp53downregulationdeterminedtheupregulationofmiR-223.(d)Mutantp53bindsmiR-223promoterinbreastcancercelllines.ChIPanalysisrevealeddirectbindingofmutantp53tothemiR-223promoterinSKBR3(breastcancercelllineexpressingp53R175H)andinMDA-MB-468.Crosslinkedandsonicatedchromatinwasimmunoprecipitatedwithanti-p53serum(5鈥壩糽sheepserumAb7JA1308;Calbiochem/Millipore)orwithamixofproteinA/G-agarosebeadswithoutanantibodyasnegativecontrol(noAb)asdescribedpreviously.16Afterpurification,immunoprecipitatedDNAwasanalyzedbyPCR.Theprimersusedamplifiedaregionspanningthetranscriptionstartsite(TSS)ofthepri-miR-223(鈭?05to+108)(describedinFukaoetal.23)(FW:5鈥?GCATCCAGATTTCCGTTGGCTAAC-3鈥?RW:5鈥?GCAAATGGATACCATACCTGTCAGTG-3鈥?.AheterochromatinregionofthegeneHist1H2BAwasusedasaninternalcontrol(hTSH2Bprimers;Diagenodesa,Li猫ge,Belgium).ThesameimmunoprecipitatedDNAwasalsoanalyzedbyreal-timePCRusingcostumedPrimetimeqPCRProbeandPrimers(IdT,Coralville,IA,USA;probe:FAM/5鈥?AGGGCAGTG-3鈥?ZEN/5鈥?GCCTGCTACTATTGCT-3鈥?3IABkFQFWprimer:5鈥?TCTGTTGAAGGTCAGCTGGGAGTT-3鈥?RWprimer:5鈥?TGCTGTTGTGAAAGGGTCTGCTAC-3鈥?designedinthesameregionasabove(鈭?05to+108).Analysisofthedatawasperformedbythepercentofinputmethod,usingthegeneHist1H2BAasaninternalcontrolgene.TheenrichmentoftheChIPsampleswithnoantibodywaszero.(e)Mutantp53inhibitsmiR-223promoteractivityinSW480cells.Aluciferasereporterconstruct(ppri-hmiR-223鈭?69-Luc)containingthemiR-223regulatoryregion(鈭?69to+91,23akindgiftformDrTaroFukao)wastransfectedinSW480,wheremutantp53wasstablysilenced(sh53)orinthescrambledcontrolcells(shSC)asin(b).Cellswereco-transfectedwitha尾-gal-expressingplasmidandthe尾-galassaywasusedforstandardization.Decreasedexpressionofmutantp53determinedhighermiR-223promoteractivity(n=6卤s.e.m.,*P-value0.002)(leftpanel).H1299cellswereco-transfectedwithaplasmidexpressingmutantp53R175Hortheemptyvector(mock)andtheppri-hmiR-223鈭?69-Lucreporterplasmid.23pRL-TKreporter(Promega,Madison,WI,USA),constitutivelyexpressingtheRenillaluciferase,wasincludedfornormalization.After48鈥塰,cellswerelysedandassayedforluciferaseactivityusingtheDualLuciferasekit(Promega)tomeasurebothRenillaandFireflyluciferaseactivities.Overexpressionofmutantp53decreasedmiR-223promoteractivity(rightpanel).