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Intactp53-DependentResponsesinmiR-34–DeficientMiceCarlaP.
Concepcion1,2,Yoon-ChiHan1,PingMu1,2,CiroBonetti1,EvelynYao1,2,AlecoD'Andrea1,JoanaA.
Vidigal1,WilliamP.
Maughan1,2,PaulOgrodowski1,AndreaVentura1*1CancerBiologyandGeneticsProgram,MemorialSloan-KetteringCancerCenter,NewYork,NewYork,UnitedStatesofAmerica,2WeillCornellGraduateSchoolofMedicalSciences,CornellUniversity,NewYork,NewYork,UnitedStatesofAmericaAbstractMicroRNAsbelongingtothemiR-34familyhavebeenproposedascriticalmodulatorsofthep53pathwayandpotentialtumorsuppressorsinhumancancers.
Toformallytestthesehypotheses,wehavegeneratedmicecarryingtargeteddeletionofallthreemembersofthismicroRNAfamily.
WeshowthatcompleteinactivationofmiR-34functioniscompatiblewithnormaldevelopmentinmice.
Surprisingly,p53functionappearstobeintactinmiR-34–deficientcellsandtissues.
AlthoughlossofmiR-34expressionleadstoaslightincreaseincellularproliferationinvitro,itdoesnotimpairp53-inducedcellcyclearrestorapoptosis.
Furthermore,incontrasttop53-deficientmice,miR-34–deficientanimalsdonotdisplayincreasedsusceptibilitytospontaneous,irradiation-induced,orc-Myc–initiatedtumorigenesis.
WealsoshowthatexpressionofmembersofthemiR-34familyisparticularlyhighinthetestes,lungs,andbrainsofmiceandthatitislargelyp53-independentinthesetissues.
ThesefindingsindicatethatmiR-34playsaredundantfunctioninthep53pathwayandsuggestadditionalp53-independentfunctionsforthisfamilyofmiRNAs.
Citation:ConcepcionCP,HanY-C,MuP,BonettiC,YaoE,etal.
(2012)Intactp53-DependentResponsesinmiR-34–DeficientMice.
PLoSGenet8(7):e1002797.
doi:10.
1371/journal.
pgen.
1002797Editor:H.
LeightonGrimes,CincinnatiChildren'sHospitalMedicalCenter,UnitedStatesofAmericaReceivedJanuary11,2012;AcceptedMay15,2012;PublishedJuly26,2012Copyright:2012Concepcionetal.
Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwassupportedbyfundsfromtheGeoffreyBeeneCancerResearchFoundation(AV),theAmericanItalianCancerResearchFoundation(CB),theLeukemiaLymphomaSociety(Y-CH),andtheGabrielleAngel'sFoundation(AV).
EYwassupportedbyanNIH-T32traininggrant.
Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:venturaa@mskcc.
orgIntroductionThetumor-suppressorproteinp53isamasterregulatorofthestressresponseandprovidesakeybarriertocellulartransforma-tionandtumorigenesis[1].
Upononcogeneactivation,DNAdamage,andotherformsofcellularstress,p53accumulatesinthenucleuswhereitinducesorrepressesthetranscriptionofamyriadofgenes.
Ultimately,p53activationresultsincellcyclearrest,apoptosis,orsenescence,dependingonthecellularcontextandthetypeofstimulus[2].
Althoughtranscription-independentmechanismshavebeenreported[3],p53mainlyactsasatranscriptionfactorforalargearrayofdownstreameffectors[4],includingtheproapoptoticproteinsPuma,Noxa,andBax,aswellasthecellcycleinhibitor,p21[5–11].
Theessentialtumor-suppressivefunctionofp53isfurtherhighlightedbytheobservationthatthispathwayisinactivatedinthevastmajorityofhumancancers[1,12].
SeveralgroupshaverecentlysuggestedthatmiRNAsarealsocomponentsofthep53pathway.
Inparticular,threehighlyrelatedmiRNAs—miR-34a,miR-34b,andmiR-34c(Figure1A)—aredirectlyinduceduponp53activationinmultiplecelltypesandhavebeenproposedtomodulatep53function[13–20].
TheprecursorsofthesemiRNAsaretranscribedfromtwodistinctloci:themiR-34alocusonchromosome1p36andthemiR-34b,clocusonchromosome11q23.
Canonicalp53-bindingsitesarelocatedinthepromoterregionsofbothmiR-34aandmiR-34b,c,andthesemiRNAsarebonafidedirecttranscriptionaltargetsofp53[13,17,18].
EctopicexpressionofmembersofthemiR-34familyissufficienttoinducecellcyclearrestorapoptosis,dependingonthecellularcontext[14,17–21].
Furthermore,loss-of-functionstudiesusingmiR-34antagonistshaveprovidedsomeevidencethatthismiRNAfamilyisrequiredforp53function[13,18,22–24].
ManyofthepredictedmiR-34targetgenesencodeforproteinsthatareinvolvedincellcycleregulation,apoptosis,andgrowthfactorsignaling.
TheseincludeCyclinE2,cMyc,MET,BCL-2,SIRT1,andmembersoftheE2Ffamilyoftranscriptionfactors[13,17,20,23,25].
Consistentwithapossibletumor-suppressorrole,lossofexpressionofmembersofthemiR-34familyhasbeenreportedinhumancancers.
HemizygousdeletionofthechromosomalregioncontainingthemiR-34alocushasbeendescribedinneuroblastomasandpancreaticcancercelllines[14,21].
Similarly,lossof11q23,containingthemiR-34b,clocus,hasbeenreportedinprostatecancers[26].
EpigeneticsilencingofmiR-34membershasalsobeenreportedinhumancancers.
Promoterhyper-methylationofmiR-34aisobservedinnon-small-celllungcancersandmelanomas[27,28],andsilencingofmiR-34aandmiR-34b,chasbeendescribedinhumanepithelialovariancancers[29].
AlthoughtheseobservationspointtowardsanimportantroleformiR-34membersascriticaldownstreameffectorsofp53andpotentialtumorsuppressors,thesehypotheseshavenotbeenformallytestedusingmiR-34-deficientanimalsandcells.
OnenotableexceptionisarecentelegantpaperbyChoiandcolleaguesdemonstratingthatmiR-34-deficientMEFsaremoresusceptiblePLoSGenetics|www.
plosgenetics.
org1July2012|Volume8|Issue7|e1002797toreprogramming[30].
However,theconsequencesofmiR-34lossonp53functionwerenotexaminedindetail.
HerewereportthegenerationofmicecarryingtargeteddeletionofallthreemembersofthemiR-34familyandsystematicallyinvestigatetheimpactofmiR-34lossonthep53pathway.
WeshowthatcompletegeneticinactivationofmiR-34doesnotdetectablyimpairthep53responseinavarietyofinvivoandinvitroassays.
Thesefindingshighlightlikelyredundanciesamongp53'sdownstreameffectors,showthatthemiR-34familyislargelydispensableforp53functioninvivo,andsuggestpossiblep53-independentfunctions.
Resultsp53-dependentandp53-independentmiR-34expressioninvivoToinvestigatethebiologicalfunctionsofmiR-34,wefirstexaminedtheexpressionofthisfamilyofmiRNAsunderbasalconditionsandinresponsetop53activationinvivo.
Underbasalconditions,miR-34aandmiR-34b,cexpressionisparticularlyintenseinthetestis,brain,andlungofadultmice(Figure1B–1D).
MiR-34b,cexpressionseemslargelyrestrictedtothesethreetissues,whilemiR-34aisdetectable,albeitatlowerlevels,alsoinavarietyofotherorgans(Figure1B–1D).
ConsistentwithpreviousreportsindicatingthatmiR-34aexpressionisunderthedirectcontrolofp53[13,17,18],wedetectedreducedlevelsofthismiRNAinasubsetofp53-deficienttissues(heart,smallandlargeintestine,liverandkidney),butthelevelsofbothmiR-34aandmiR-34b,cremainedhighinthebrains,testesandlungs(Figure1B–1D)ofp532/2mice,afindingthatsuggeststhatp53-independentmechanismsdeterminebasalmiR-34transcriptioninthesetissues.
Theseresultswereobtainedusingtwoindependenttechniques:quantitativerealtimepoly-merasechainreaction(qPCR)andNorthernblotting.
ThespecificityandsensitivityoftheseassayswerevalidatedusingmiR-34-deficientmiceascontrols(Figure1B–1DandFigure2D).
Exposuretoionizingradiation,whichleadstop53stabilizationandtranscriptionalactivation,resultedinsubstantialmiR-34ainductioninthespleen,thymus,smallandlargeintestineofwild-typemice,butnotintheothertissuesexamined(FigureS1).
WealsoobservedmodestbutsignificantmiR-34cinductioninthethymus,smallandlargeintestineofirradiatedmice,butnotintheothertissuesexamined.
GenerationofmiR-34-deficientmiceToinvestigatethephysiologicfunctionsofthemiR-34familyandtodeterminetheextenttowhichitsinductionisrequiredforp53function,wegeneratedmicecarryingtargeteddeletionofbothmiR-34aandmiR-34b,cloci(Figure2A–2C).
Toallowtemporallyandspatiallyrestricteddeletion,wealsogeneratedaconditionalmiR-34aKOallele(miR-34afl,Figure2A).
NorthernblotandqPCRanalysisconfirmedthelossofexpressionoftherespectivemiRNAsinhomozygousmutantanimals(Figure2D).
Importantly,homozygousdeletionofmiR-34adidnotleadtocompensatoryup-regulationofmiR-34b,c,andviceversa(Figure2Danddatanotshown).
MiR-34a2/2andmiR-34b,c2/2singleKOmicewereviableandfertileandwereobtainedattheexpectedMendelianfrequency(Figure2E,2F).
ThesequencesimilaritybetweenthethreemiR-34familymembers(Figure1A),whichsharethesame''seed'',suggeststhattheymaybefunctionallyredundant.
ToexaminetheconsequencesofcompletelossofmiR-34function,wecrossedmiR-34a2/2andmiR-34b,c2/2micetogeneratecompoundmutantanimalscarryinghomozygousdeletionofallthreefamilymembers(miR-34TKO/TKO).
CompletelossofmiR-34expressioninmiR-34TKO/TKOanimalswasconfirmedbyNorthernblotandqPCR(Figure2D).
MiR-34TKO/TKOmiceofbothsexeswereobtainedatapproximatelytheexpectedMendelianfrequency(Figure2G),didnotdisplayobviousmacroscopicdefects(FigureS2),andwerefertile(datanotshown).
Afullhistologicalexamination(FigureS3),completebloodcellcount(FigureS4),andserumchemistryanalysis(FigureS5)didnotdetectanystatisticallysignificantdefectsinadultmiR-34TKO/TKOmiceofbothsexes.
Ananalysisofthemajormyeloidandlymphoidpopulationsofthebonemarrow,spleenandthymusalsodidnotrevealanystatisticallysignificantdifferencebetweenwild-typeandmiR-34TKO/TKOmice(FigureS6).
P53-dependentcellcyclearrestinmiR-34TKO/TKOMEFsNext,wesoughttodeterminewhetherlossofmiR-34expressionaffectsthep53responseinvitro.
Wefocusedonthethreebest-characterizedp53-dependentprocesses:replicativesenescence,responsetoDNAdamage,andresponsetooncogeneactivation[31–35].
Theabilitytoproliferateindefinitelyisoneofthehallmarksofcancercells[36]andalsooneofthemoststrikingconsequencesofp53inactivationatthecellularlevel[35].
ToinvestigatetheroleofmiR-34inreplicativesenescence,mouseprimaryfibroblasts(MEFs)derivedfromwild-type,p532/2,andmiR-34TKO/TKOembryoswereseriallypassaged.
Althoughwedetectedaremark-ableinductionofmiR-34aandmiR-34cexpressioninlate-passagewild-typeMEFscomparedtoearly-passageMEFs(Figure3A),miR-34-deficientMEFsbecamesenescentwithakineticidenticaltowild-typeMEFs(Figure3B).
Thisisinstarkcontrastwithp53-deficientMEFs,whichasexpectedproliferatedindefinitely(Figure3B).
TheonlysignificantdifferenceweobservedwasaslightbutreproducibleincreaseintheproliferationrateofearlypassagemiR-34-deficientfibroblastscomparedtocontrols(Figure3B,3C).
WenextexaminedtheroleofmiR-34intheresponsetotheDNAdamagingagentdoxorubicin.
Aspreviouslyreported[37],doxorubicintreatmentleadstostabilizationofp53(Figure3D)andup-regulationofitsdownstreamtargetsp21(Cdkn1a),Mdm2,PumaandNoxa(Figure3D–3F).
ExpressionofmembersofthemiR-34familywassimilarlyupregulatedinresponsetop53stabilization(Figure3G).
Althoughaspredicted,p53-nullcellsAuthorSummaryMicroRNAs(miRNAs)aresmall,non-codingRNAsthatbroadlyregulategeneexpression.
MicroRNAderegulationisacommonfeatureofhumancancers,andnumerousmiRNAshaveoncogenicortumorsuppressiveproperties.
MembersofthemiR-34family(miR-34a,miR-34b,andmiR-34c)havebeenwidelyspeculatedtobeimportanttumorsuppressorsandmediatorsofp53function.
Despitethegrowingbodyofevidencesupportingthishypothesis,previousstudiesonmiR-34havebeendoneinvitroorusingnon-physiologicexpressionlevelsofmiR-34.
Here,weprobethetumorsuppressivefunctionsofthemiR-34familyinvivobygeneratingmicecarryingtargeteddeletionoftheentiremiR-34family.
OurresultsshowthatthemiR-34familyisnotrequiredfortumorsuppressioninvivo,andtheysuggestp53-independentfunctionsforthisfamilyofmiRNAs.
Importantly,themicegeneratedfromthisstudyprovideatoolforthescientificcommunitytofurtherinvestigatethephysiologicfunctionsofthemiR-34family.
miR-34-NullMiceandp53PLoSGenetics|www.
plosgenetics.
org2July2012|Volume8|Issue7|e1002797failedtoarrestinG1inresponsetodoxorubicintreatment,theresponseofmiR-34TKO/TKOMEFswasindistinguishablefromthatofwild-typecells(Figure3H–3I).
Consistentwiththeseresults,doxorubicintreatmentcausedsimilaractivationofp53andofitsdownstreamtargetsinwild-typeandmiR-34TKO/TKOMEFs(Figure3Eand3F).
Theexperimentsdescribedabovewereperformedonasyn-chronouslygrowingearly-passageMEFsandassuchmaynotbesensitiveenoughtodetectamodesteffectofmiR-34lossontheS-phasecheckpoint.
Tomeasurecellcycleprogressionmoreaccurately,wefirstsynchronizedMEFsbyserumstarvationandthenreleasedthecellsincompletemediumcontainingcolcemid,amitoticspindleinhibitor.
Withthisexperimentaldesign,uponreleaseincompletemedium,cellssynchronouslyproceedfromG1throughSphaseandthenaccumulateattheM(4N)phase.
ThisassayprovidesamoresensitivewaytodeterminetheabilityofcellstotransitionthroughtheS-phaseandallowsdetectionofsubtledefectsintheDNAdamage-inducedS-phasecheckpoint.
AlthoughareproduciblylargerfractionofmiR-34TKO/TKOcellswasabletotransitionthroughtheSphaseafterionizingradiationcomparedtowild-typeMEFs(Figure3J),weobservedasimilardifferenceinnon-irradiatedMEFs(Figure3J).
ThemostlogicalinterpretationoftheseresultsisthatmiR-34-deficientMEFs,ratherthanbeingmoreresistanttoirradiation-inducedcellFigure1.
MiR-34expressioninwild-typeandp532/2mousetissues.
(A)SequencealignmentofmousemiR-34a,miR-34bandmiR-34c.
Differingnucleotidesarecoloredinblue.
Theseedsequencesareinbold.
(B–D)MiR-34aandmiR-34cexpressionasdetectedbyqPCR(B,C)andbyNorthernblotting(D)intissuesofwild-typeandp532/2mice.
doi:10.
1371/journal.
pgen.
1002797.
g001miR-34-NullMiceandp53PLoSGenetics|www.
plosgenetics.
org3July2012|Volume8|Issue7|e1002797cyclearrest,possessaslightlyfasterbasalproliferationormorerapidre-entryintothecellcyclefollowingserumstarvation.
ThisinterpretationisalsoconsistentwiththefasterproliferationratedisplayedbymiR-34-deficientMEFs(Figure3B,3C)andwiththeobservationbyLalandcolleaguesthatmiR-34aisinvolvedinmodulatingthecellularresponsetogrowthfactors[38].
WealsoexaminedtheconsequencesofmiR-34lossinMEFsontheexpressionofasubsetofitspreviouslyreporteddirecttargets[17,20,23,25].
WedetectedmodestupregulationofcMyc,E2f3,MetandSirt1inmiR-34-deficientMEFs,whileBcl2wasexpressedatsimilarlevelsinwild-typeandmutantcells(Figure3K).
TheupregulationofMycandE2f3mightcontributetotheincreasedproliferationratewehaveobservedinmiR-34deficientMEFs.
P53-dependentapoptosisinmiR-34TKO/TKOcellsandmiceHavingestablishedthatmiR-34isnotrequiredforcellcyclearrestinresponsetogenotoxicstressinMEFs,wenextsoughttodeterminewhetherthismiRNAfamilymightcontributetop53-inducedapoptosis.
Thymocytesrespondtoionizingradiationsbyrapidlyundergoingapoptosis,aneffectthatisdependentonp53[39].
WethereforeexaminedtheeffectsofDNAdamageonthymocytesfromwild-type,p532/2,andmiR-34TKO/TKOmice.
Asexpected,p532/2thymocyteswerealmostentirelyresistanttoirradiation-inducedapoptosis;however,wild-typeandmiR-34-deficientcellswereequallysensitivetoDNAdamage-inducedapoptosis,asjudgedbydose-responseandtime-courseexperiments(Figure4A,4B).
ToexcludethepossibilitythattissuecultureconditionsmayhavemaskedaphysiologicroleofmiR-34inmodulatingthep53response,wenextexaminedtheconsequencesofp53activationinmiR-34-deficienttissuesdirectlyinvivo.
Age-andsex-matchedwild-type,miR-34TKO/TKOandp532/2micewereexposedto10Gyofionizingradiationandeuthanized6hourslater.
Ionizingradiationinducedsimilaractivationofthep53pathwayandofitsdownstreameffectorsinwild-typeandmiR-34TKO/TKOmice(Figure4C).
Analogoustowhatweobservedinthymocytesinvitro,theapoptoticresponsewasequallydramaticinwild-typeandinmiR-34-deficientmice,whileitwasvirtuallyabsentinp532/2animals(Figure4D–4G).
Figure2.
TargeteddeletionofmiR-34aandmiR-34b,c.
(A)TargetingandscreeningstrategyforthegenerationofconstitutiveandconditionalmiR-34aKOalleles.
TherestrictionsitesusedfortheSouthernblotscreeningareindicated(S=SphI,E=EcoRI).
Thegraybarwithanasteriskrepresentsagenomicregionabsentinthe129SvJaestrainbutpresentintheC57BL/6strain,whichresultsintwodistinctsizesindigestions.
(B)TargetingandscreeningstrategyforthegenerationofmiR-34b,cKOallele(H=HindIII,S=SpeI).
(C)GenotypingbytailgenomicPCRshowinggermlinetransmissionofthemiR-34adeletedandfloxedalleles(upperpanel),andthemiR-34b,cdeletedallele(lowerpanel).
(D)Northernblotting(upperpanel)ontotalRNAextractedfromthetestesofmicewiththeindicatedgenotypes.
ProbesspecificformiR-34aandmiR-34cwereused.
CompletelossofmiR-34aandmiR-34cexpressionwasfurtherconfirmedinMEFsbyqPCR(lowerpanel).
RepresentativepicturesofmiR-34a2/2(E),miR-34b,c2/2(F),andmiR-34TKO/TKO(G)malesat4weeksofage.
Thetablebeloweachpicturesummarizestheexpectedandobservedfrequenciesofmiceofeachgenotypeasobtainedfromheterozygousinter-crosses.
ForthemiR-34TKOallele(G),doubleheterozygousmicewereinter-crossed.
doi:10.
1371/journal.
pgen.
1002797.
g002miR-34-NullMiceandp53PLoSGenetics|www.
plosgenetics.
org4July2012|Volume8|Issue7|e1002797Figure3.
Responsetop53activationinmiR-34TKO/TKOmouseembryonicfibroblasts(MEFs).
(A)MiR-34aandmiR-34cexpressioninserially-passagedwild-typeMEFs,asmeasuredbyqPCR.
Errorbarsindicate1standarddeviation(SD).
(B)Cumulativepopulationdoublingsofwild-type,miR-34TKO/TKOandp532/2MEFs.
Errorbarsindicate1SD.
(C)Growthcurvesofwild-typeandmiR-34TKO/TKOMEFs.
Errorbarsindicate1SD.
(D)Immunoblotsofp53,p21andMdm2inwild-type(W)andmiR-34TKO/TKO(K)MEFstreatedwith0.
2mg/mldoxorubicinfortheindicatedtime.
(E)Expressionofselectedp53targetsintotalRNAfromdoxorubicin-treatedMEFs.
Cellsweretreatedwith0.
2mg/mldoxorubicinfor12hours(Dox)orleftuntreated(U).
ExpressionoftheindicatedgeneswasdeterminedbyqPCR.
Errorbarsrepresent1SD.
(F)Immunoblotsshowingp53activationinthreewild-typeandthreemiR-34TKO/TKOMEFlines.
Cellswereleftuntreatedortreatedwith0.
2mg/mldoxorubicinfor12hours.
(G)TimecourseofmiR-34aandmiR-34cexpressioninwild-typeandp532/2cellstreatedwith0.
2mg/mldoxorubicin.
MicroRNAexpressionwasdeterminedbyqPCR.
Errorbarsindicate1SD.
(H,I)Cellcycledistributionofwild-typeandmiR-34TKO/TKOMEFs.
AsynchronouslygrowingMEFsoftheindicatedgenotypeweretreatedwithincreasingdosesofdoxorubicinfor16hours(H),orwith0.
2mg/mldoxorubicinforincreasingtime(I).
Errorbarsindicate1SD.
(J)Upperpanel:cellcycledistributionofwild-type,miR-34TKO/TKO,andp532/2MEFsafter72hoursinstarvationmedium(grayhistogram).
Starvedcellswerereleasedincompletemediumcontainingcolcemidandmock-treated(lightbluehistogram)orexposedto20Gyirradiation(redhistogram).
Cellswereanalyzedby7-AADstainingattheindicatedtimeafterreleaseincompletemedium.
Lowerpanel:percentagesofirradiatedanduntreatedmiR-34-NullMiceandp53PLoSGenetics|www.
plosgenetics.
org5July2012|Volume8|Issue7|e1002797BasedontheseresultsweconcludethatmiR-34functionisnotrequiredforp53-inducedcell-cyclearrestandapoptosisinresponsetogenotoxicstresses.
miR-34andtumorsuppressioninvitroThep53pathwayprovidesacrucialbarrieragainsttheneoplastictransformationofprimarycells[40].
Supra-physiologicproliferativestimuli,suchasthosecausedbysustainedoncogeneactivation,leadtotranscriptionalactivationofp19Arf,whichinturnresultsinstabilizationandactivationofp53,andconsequentlyapoptosisorcellcyclearrest[41].
Forexample,ectopicexpressionofaconstitutivelyactiveK-Ras(K-RasV12)inwild-typeMEFsleadstooncogene-inducedsenescence,buttheconcomitantinactivationofp53issufficienttoallowfullcellulartransformation[31].
TotestwhethermiR-34playsaroleinthiscontext,weectopicallyexpressedoncogenicK-Rasinwild-type,miR-34TKO/TKO,andp532/2MEFs.
AsshowninFigure5A,completelossofmiR-34functionwasnotsufficienttoallowprimaryMEFstobetransformedbyK-RasV12alone,whilep53-deficientMEFswerereadilytransformedinthesameassay.
However,whenMEFswereco-transducedwithoncogenicK-RasandE1A,whichbindstoandinhibitstheretinoblastomaprotein(pRb)[42],weobservedaslightincreaseinthenumberoffociformedinmiR-34TKO/TKOMEFscomparedtowild-typecells(Figure5A,5B).
TheseresultsshowthatwhilemiR-34aloneisnotrequiredforp53-mediatedtumorsuppressioninMEFs,itslossmightcooperatewithinactivationoftheRbpathwayinpromotingcellulartransformation.
miR-34andtumorsuppressioninvivoToextendouranalysistoaninvivosetting,wenextexaminedwhethermiR-34inactivationissufficienttoacceleratespontaneousandoncogene-inducedtransformationinmice.
P53-deficientmiceexhibitahighincidenceofspontaneoustumors,inparticularlymphomasandsarcomas[43–45],andp53inactivationgreatlyacceleratestumorformationinavarietyofmousemodelsofhumancancer[46–51].
TodeterminewhetherlossofmiR-34expressionleadstoincreasedspontaneoustumorigenesis,weagedacohortof14miR-34TKO/TKOand12wild-typemice.
Theanimalsweremonitoredforatleast12months(wild-type=359days;miR-34TKO/TKO=359days)andupto17.
3months(wild-type=521days;miR-34TKO/TKO=521days).
Allwild-typeandmiR-34TKO/TKOmiceappearedhealthyandmiR-34TKO/TKOmicedidnotshowareductioninlifespancomparedtowild-typecontrols(FigureS7).
Forcomparison,themediansurvivalofp532/2micehasbeenreportedtobe4.
5monthsandby10monthsofageallp532/2micehavediedordevelopedtumors[45].
Inaddition,,40%ofp53+/2micedeveloptumorsby16monthsofage[45].
Thus,althoughalongerfollow-upofmiR-34TKO/TKOmicemaybeneededtouncoververysubtledefectsintumorsuppression,weconcludethatlossofmiR-34expressiondoesnotleadtoasubstantialincreaseinspontaneoustumorigen-esis.
WenextsoughttodeterminewhetherlossofmiR-34mightacceleratetumorformationinresponsetogenotoxicstress.
P532/2miceirradiatedshortlyafterbirthdisplayacceleratedtumori-genesiscomparedtonon-irradiatedlittermates[52].
Wethereforeexposedacohortof14miR-34TKO/TKOand11wild-typemiceto1Gyofionizingradiationsoonafterbirthandmonitoredthemfor42–60weeks.
Bothwild-typeandmiR-34-deficientmiceappearedhealthythroughoutthefollow-upperiod(FigureS7),instrikingcontrastwiththe,15weeksreportedmediantumor-freesurvivalofirradiatedp532/2mice[52].
Althoughitwillbeimportanttofollowalargercohortofanimalsoveramoreprolongedperiod,theseresultssuggestthatmiR-34doesnotprovideapotentbarriertotumorigenesisinresponsetogenotoxicstressinvivo.
Finally,wesoughttodeterminewhethergeneticablationofmiR-34couldcontributetotumorformationincooperationwithadefinedoncogeniclesion.
Fortheseexperiments,wechosetheEm-MycmodelofBcelllymphomas[53].
Acrucialtumor-suppressiveroleforp53iswellestablishedinthismousemodelandinactivationofthep53pathwayresultsingreatlyacceleratedlymphomagenesis[46,47,54].
However,eveninthiscontextcompletelossofmiR-34expressionwasnotsufficienttoacceleratetumorformation.
TheincidenceandlatencyofBcelllymphomaswasvirtuallyidenticalinEm-Myc;miR-34TKO/TKOandEm-Myc;miR-34+/+mice(Figure5C)andtheresultingtumorsdisplayedsimilarhistopathologicalfeaturesandextentofsponta-neousapoptosis(Figure5D–5E).
DiscussionWehavereportedthegenerationofmicecarryingtargeteddeletionofmiR-34a,miR-34bandmiR-34c,andwehaveinvestigatedtheconsequencesoflossofmiR-34expressiononp53-dependentresponsesinvitroandinvivo.
OurresultsshowthatcompletelossofmiR-34expressioniscompatiblewithnormaldevelopmentandthatthep53pathwayisapparentlyintactinmiR-34-deficientmice.
OurobservationthatinactivationofmiR-34doesnotimpairp53-mediatedresponsesinvitroandinvivoisparticularlyrelevantbecauseakeyroleformiR-34inthep53pathwayhadbeenpreviouslyproposedbyanumberofindependentgroups.
TheresultspresentedinthispaperdonotnecessarilyconflictwithpreviousexperimentsshowingthatectopicexpressionofmiR-34caninducemanyofthemostcharacteristicconsequencesofp53activation;herewehavetestedwhethermiR-34isnecessaryforp53functionandnotwhetheritissufficient.
Moredifficult,however,istoreconcileourfindingswithpreviousreportsofimpairedp53-functionincellstreatedwithmiR-34antagonists.
BecausepreviousworkhasreliedontheuseofmiRNAantagoniststoinhibitmiR-34function,itispossiblethatsomeofthepreviousobservationsreflectedmiR-34-indepen-dentoff-targeteffects.
ItisalsopossiblethatothermiRNAssharingsequencesimilaritieswithmiR-34maycompensateformiR-34lossintheknock-outanimals.
Inparticular,membersofthemiR-449family(miR-449a,bandc)havethesame''seed''sequenceasmiR-34,andmiR-34antagonistscouldinprincipleimpairtheirfunctionaswell.
AconclusivetestforthishypothesiswillrequirethegenerationofcompoundmiR-34andmiR-449mutantanimals,butseverallinesofevidencesuggestthatthisexplanationisnotparticularlylikely.
First,inthetissuesandcellsusedinourexperiments,theexpressionofmiR-449membersismuchlowercomparedtomiR-34aandmiR-34c,asjudgedbymultipleindependentmethodsincludingqPCR,Northernblottingandhighthroughputsequencing(FigureS8anddatanotshown).
Anotableexceptionisrepresentedbythetestis,inwhichexpressioncellsinG1andG2-Mphasesafter24hoursincompletemedium.
Experimentswereperformedonthreeindependentwild-typeandthreeindependentmiR-34TKO/TKOMEFlines.
(K)ImmunoblotdetectionofpredictedmiR-34targetsonthreeindependentwild-typeandthreeindependentmiR-34TKO/TKOMEFlines.
doi:10.
1371/journal.
pgen.
1002797.
g003miR-34-NullMiceandp53PLoSGenetics|www.
plosgenetics.
org6July2012|Volume8|Issue7|e1002797Figure4.
p53-dependentapoptosisinthymocytesandinvivo.
(A)Percentagesofviablewild-type,miR-34TKO/TKO,andp532/2thymocytes16hoursaftertreatmentwithincreasingdosesofirradiation(0,2,4,6,8,and10Gy).
Errorbarsrepresent1SD.
(B)Percentagesofviablewild-type,miR-34TKO/TKO,andp532/2thymocytes4,8,and24hoursafterirradiation(5Gy).
Errorbarscorrespondto1SD.
(C)Expressionlevelsofp53transcriptionaltargetsinthethymiandspleensofuntreated(U)andirradiated(IR,10Gy)wild-type,miR-34TKO/TKOandp532/2micebyqPCR.
(D,E)Representativecleavedcaspase-3immunohistochemistryofthethymus(D)andthesmallintestine(E)ofuntreatedandirradiated(10Gy)wild-type,miR-34TKO/TKOandp532/2mice(n=3micepergroup).
Brownstainingindicatescleavedcaspase-3(CC3).
(F,G)Quantificationofapoptosisinthethymus(F)andintheintestine(G)ofcontrolandirradiatedanimals.
InpanelFtherelativestainingintensityaveragedoverthreemicroscopicfieldspersampleisplotted.
InpanelG,theaveragenumberofCC3-positivecellspercryptisplotted.
Atleast25randomlyselectedcryptspersamplewerecounted.
Errorbarscorrespondto1SD.
Pvalueswerecalculatedusingthetwo-tailedStudent'st-test.
doi:10.
1371/journal.
pgen.
1002797.
g004miR-34-NullMiceandp53PLoSGenetics|www.
plosgenetics.
org7July2012|Volume8|Issue7|e1002797ofmiR-449aisparticularlyelevated(FigureS8).
Inaddition,miR-449expressionisnotsubstantiallyincreasedinmiR-34-nullmice,andactivationofthep53pathwaydoesnotleadtosignificantupregulationofmiR-449(FigureS8).
WewouldliketoemphasizethatourresultsdonotnecessarilyindicatethatmembersofthemIR-34familyarenotcomponentsofthep53pathway.
Giventheessentialtumor-suppressivefunctionexertedbyp53,itisperhapsnotsurprisingthatmultipleFigure5.
Oncogene-inducedtransformationinmiR-34TKO/TKOfibroblastsandmice.
(A)Representativefocusformationassaysofwild-type,miR-34TKO/TKO,andp532/2MEFs.
MEFSwereinfectedwithretrovirusesexpressingK-RasV12aloneorK-RasV12andE1A.
Theresultsarerepresentatitveoftwoindependentexperimentsperformedonatotaloffourwild-typeandfourmiR-34TKO/TKOMEFlines.
(B)Barplotshowingthenumberoftransformedfoci.
Errorbarsare1SD.
(C)SurvivalcurvesofEm-Myc;miR-34+/+andEm-Myc;miR-34TKO/TKOmice.
P-valuewascalculatedusingthelog-rank(Mantel-Cox)test.
(D)Histopathologyandcleavedcaspase-3(CC3)immunohistochemistryofrepresentativelymphomasobtainedfromEm-Myc;miR-34+/+andEm-Myc;miR-34TKO/TKOmice.
(E)BarplotshowingthenumberofCC3-positivecellsperlowmagnificationfield.
FiveEm-Myc;miR-34+/+tumorsandandfourEm-Myc;miR-34TKO/TKOtumorswereanalyzed.
Errorbarsindicate1SD.
doi:10.
1371/journal.
pgen.
1002797.
g005miR-34-NullMiceandp53PLoSGenetics|www.
plosgenetics.
org8July2012|Volume8|Issue7|e1002797andpartiallyredundanteffectorarmsarerecruitedinresponsetoitsactivation.
Itisplausiblethatthesimultaneousinactivationofmultipleeffectorarmsisrequiredtomeasurablyimpairp53function.
ConsistentwiththismodelisourobservationthatwhilelossofmiR-34expressionalonedoesnotallowthetransformationofprimarycellsbyoncogenicK-Ras,itslightlyincreasestheefficiencyoftransformationwhencombinedwithinactivationoftheRbpathwaybyE1A(Figure5A,5B).
Inthiscontext,itwillbeimportanttosystematicallyprobetheextentoffunctionalcooperationbetweenthisfamilyofmiRNAsandother,previouslycharacterizedp53effectors.
Wealsowishtopointoutthatinthismanuscriptwehaveinvestigatedthebest-characterizedfunctionsofp53(cellcyclearrest,apoptosisandtumorsuppression)anditremainspossiblethatmiR-34participatesinotherp53-dependentprocesses.
Forexample,p53hasbeenproposedtomodulateautophagy[55]andstemcellquiescence[56,57]andwecannotexcludethatmiR-34playsanimportantroleinthesecontexts.
FuturestudiesusingthemiR-34-deficientanimalswehavegeneratedwillbeneededtotestthesepossibilities.
WithrespecttothepotentialtumorsuppressiveroleofmiR-34,ourexperimentsindicatethatlossofmiR-34expressiondoesnotleadtoanobviousincreaseintumorincidenceinmiceanddoesnotcooperatewithMycinthecontextofBcelllymphomagenesis.
However,thetumorsuppressivefunctionofmiR-34mightberestrictedtospecifictissuesandlossofmiR-34mightcooperatewithspecificoncogeniclesions.
Inhumans,forexample,lossofmiR-34expressionhasbeenreportedinalargefractionofprimarymelanomas,prostaticadenocarcinomasandsmallcelllungcancers[27,28],amongothers.
IntroducingthemiR-34-nullalleleswehavegeneratedintomousemodelsofthesetypesofhumancancerswillbeimportanttofullyexplorethetumorsuppressivepotentialofthisfamilyofmiRNAs.
Anadditionalissueraisedbytheresultspresentedinthismanuscriptrelatestopossiblep53-independentfunctionsofmiR-34.
WeshowthatunderbasalconditionstheexpressionofbothmiR-34lociisparticularlyelevatedinthetestesand,toalesserextent,inthebrainsandlungsofmice.
Importantly,inthesethreetissues,miR-34expressionisalmostentirelyp53-independent(Figure1B–1Dand[58]),afindingthatsuggeststhatadditionaltranscriptionfactorscontroltheexpressionofthisfamilyofmiRNAsintheabsenceofgenotoxicoroncogenicstresses.
AroleformiR-34cinspermatogenesisandincontrollingthefirstzygoticcleavagehasbeenrecentlyproposed[58,59].
AlthoughourobservationthatsingleKOandmiR-34TKO/TKOmiceproduceviableoffspringarguesagainstanessentialroleformiR-34intheseprocesses,membersoftherelatedmiR-449family,thatareparticularlyhighlyexpressedinthetestis(FigureS8),couldpartiallycompensateformiR-34lossinthiscontext.
RecentreportshavealsoimplicatedmiR-34inneuronaldevelopmentandbehavior[60,61]andaroleformiR-34cinlearningandmemory[62],aswellasinstress-inducedanxiety[63],hasbeenreported.
Inaddition,inactivationofmiR-34expressionhasbeenrecentlyshowntoleadtoacceleratedneurodegenerationandageinginDrosophilamelanogaster[64].
Adetailedbehavioralandneuroanatomicalanalysis,aswellasacarefulcharacterizationofthelong-termconsequencesofmiR-34-losswillbeessentialtoconfirmandextendthesehypothesesinmice.
Inconclusion,wehavereportedthegenerationandcharacter-izationofmiR-34-deficientmicewithaparticularfocusontheconsequencesofmiR-34lossonthep53pathway.
ThegeneticallyengineeredmousemodelsdescribedinthisstudywillbeessentialtofurtherinvestigatethephysiologicfunctionsandthetumorsuppressivepotentialofthisimportantmiRNAfamily.
MaterialsandMethodsGenerationofmiR-34constitutiveandconditionalknockoutmiceThe''recombineering''method[65]wasusedtomodifyaBACclone(RP-23-410P10)containingthemiR-34alocustogeneratethemiR-34aconditionalknockoutallele.
Afrt-Neo-frt-loxPcassettewasfirstinserted,480bpdownstreamofthepre-miR-34asequence.
Gap-repairwasusedtoretrievea9.
6kbpfragmentcontainingthefrt-Neo-frt-loxPcassette,,4kbof39homologyarm,and,3.
7kb59homologyarm,andincludingthepre-miR-34asequence.
ThefragmentwasclonedintothetargetingplasmidpKS-DTA,andasecondloxPsitewasintroducedintoauniqueKpnIsitelocated,500bpupstreamofthepre-miR-34asequence.
ThefinaltargetingconstructwaslinearizedwithNotIandelectroporatedintoV6.
5murineembryonicstemcells(ESC).
FollowingselectionwithG418,ESCcolonieswereisolatedandscreenedbySouthernblottingusingDNAprobesmappingoutsidethetargetedregion.
TwotargetedcloneswereexpandedandinjectedintoC57BL/6blastocyststogeneratechimericmice.
HighcontributionchimerasweresubsequentlycrossedtoActin-flpetransgenicmice[66]toexcisethefrt-Neo-frtcassetteandgeneratethemiR-34aconditionalknockoutallele(miR-34afl)orcrossedtoCAG-Cremice[67]toexcisetheentireregionflankedbytheloxPsitesandobtaintheconstitutivemiR-34aKOallele(miR-34aD).
Lastly,miR-34a+/flandmiR-34a+/2wereinter-crossedtoobtainmiR-34afl/flandmiR-34a2/2animals.
TogeneratemicecarryingdeletionofthemiR-34b,cbicistroniccluster,weusedrecombineeringtoreplacea1.
3kbpDNAregioninBACRP-23-281F13containingpre-miR-34bandpre-miR-34cwithafrt-Neo-frtcassette.
A8.
4kbpDNAfragmentcontainingthefrt-Neo-frtcassette,the3.
7kbp59homologyarm,and2.
8kbpof39homologyarmwasretrievedfromtheengineeredBACandclonedintopKS-DTA.
TheresultingtargetingvectorwaslinearizedbyNotIandelectroporatedintoV6.
5ESCs.
Uponselection,twoindependentcloneswereinjectedintoC57BL/6blastocysts.
HighcontributionchimeraswerecrossedtoActin-flpetransgenicmiceforgermlinetransmissionofthetargetedalleleandtodeletetheNeocassetteresultinginthemiR-34b,cDallele.
ThemiR-34b,c+/2micewereintercrossedtoobtainmiR-34b,c2/2animals.
TheEm-MycmiceweregeneratedanddescribedbyAdamsandcolleagues[53]andthep532/2miceweregeneratedbyJacksandcolleagues[44].
GenotypingprotocolsareprovidedinTextS1.
AllanimalstudiesandprocedureswereapprovedbytheMSKCCInstitutionalAnimalCareandUseCommittee.
Miceweremaintainedinamixed129SvJaeandC57BL/6background.
ThemiR-34a,floxed.
miceandthemiR-34b,c2/2miceareavailabletotheresearchcommunitythroughTheJacksonLaboratory(JAXStockNumbers018545and018546).
GenerationofMEFsPrimaryMEFlinesweregeneratedfromE13.
5embryosusingstandardprotocols.
miR-34TKO/TKOembryoswereobtainedbyintercrossingmiR-34mutantmice.
Wild-typeMEFsweregeneratedinparallel.
p532/2embryoswereobtainedbyintercrossingp53+/2mice.
GenotypingprotocolsareprovidedinTextS1.
MiR-34wild-typeandmiR-34TKO/TKOMEFlineswerealsoverifiedbyqPCR.
NorthernblottingandqPCRRNAextractionwasperformedbyhomogenizingtissuesandcellsinTRIzolreagent(Invitrogen)accordingtomanufacturer'sinstructions.
ForNorthernblotting,15mgofeachRNAsamplemiR-34-NullMiceandp53PLoSGenetics|www.
plosgenetics.
org9July2012|Volume8|Issue7|e1002797wasloadedintoa15%Urea-PAGEgelandblottedontoaHybond-N+nylonmembrane(GEHealthcare).
Theblotswerethenhybridizedwith32P-labeledprobesspecificformiR-34a,miR-34c,andU6.
qPCRwasperformedusingprimersandprobesbyAppliedBiosystemsaccordingtomanufacturer'sinstructions.
Sno-135wasusedfornormalization.
CellcultureandcellcycleanalysisPassage2or3primaryMEFswereusedforallexperimentsandculturedat37uC(5%CO2)inDME-HGwith10%FBS(completemedium)or0.
1%FBS(starvationmedium)supplementedwithL-glutamine,penicillin,streptomycin,andb-mercaptoethanol.
ForBrdUcellcycleanalysis,wild-type,miR-34TKO/TKO,andp532/2MEFswereplatedincompletemediumat70%confluence,treatedwithvaryingdosesofdoxorubicinfor16hoursortreatedatdifferenttimepoints,andpulsedwith10mMBrdUforonehour.
TheBDPharmingenAPC-BrdUkitwasusedtoprocessharvestedsamplesandusedaccordingtomanufacturer'sprotocol.
Fortheirradiationexperiments,150,000wild-type,miR-34TKO/TKOandp532/2MEFswereseededintoeachwellofa6-wellcultureplateandstarvedfor72hours.
MEFlineswerethentrypsinizedandresuspendedincompletemediumandeitherirradiated(20Gy,Cs-137irradiator,ShepherdMark-I)orleftuntreated.
Cellswerereplatedintocompletemediumcontaining500ng/mlcolcemidat70%confluenceandharvested24hlater.
Sampleswereprocessedasmentionedaboveandstainedwith7-AAD.
FlowcytometrywasperformedusingFACSCalibur(BDBiosciences),anddatawereanalyzedusingFlowJosoftware(TreeStar).
Growthcurvesand3T3assayWild-typeandmiR-34TKO/TKOMEFswereseededintoa6-wellplate(40,000cells/well)andcountedeverydayforthegrowthcurves.
Thestandard3T3protocolwasfollowedtodeterminethecumulativepopulationdoublingsofwild-type,miR-34TKO/TKO,andp532/2MEFs.
Briefly,36105cellswereseededina6cm2dishandcountedandpassagedeverythreedays.
ThymocyteapoptosisassayThymocyteswereisolatedfromsex-matched,age-matchedwild-type,miR-34TKO/TKO,andp532/2miceandseededatadensityof16106cells/mlinMEFmedium.
Thymocyteswerethentreatedwithvariousdosesofirradiation(2,4,6,8,and10Gy,Cs-137irradiator,ShepherdMark-I)orleftuntreated.
Forthetimecourseexperiments,thymocytesweretreatedwith5Gyofirradiationandharvested4,8and24haftertreatment.
SampleswerestainedwithAnnexinVandpropidiumiodide(Roche)accordingtomanufacturer'sprotocol.
FlowcytometrywasperformedusingFACSCalibur(BDBiosciences)anddatawereanalyzedusingFlowJosoftware(TreeStar).
FocusformationassayPhoenixcells(Orbigen)weretransfectedusingFUGENE6(Promega)withretroviralconstructsofK-RasV12aloneortogetherwithE1Aaccordingtomanufacturer'sinstructions.
Wild-type,miR-34TKO/TKO,p532/2MEFswereseededat70%confluenceandinfectedwithvirus.
Plateswerefixedwithmethanolandstainedwithcrystalviolettwoweeksafterinfection.
FociwerequantifiedusingImageJ.
WesternblottingandantibodiesCellswerelysedinRIPAbuffercontainingproteaseinhibitors.
Proteins(25mg)wereseparatedonaNuPAGEBis-Trisgel(Invitrogen),andtransferredontoaPVDFmembrane(Millipore).
Blockingwasperformedwith5%milkinTBST.
Primaryantibodiesusedwereanti-p21(1:1000,SantaCruz,F-5),anti-Mdm2(1:1000,Abcam,2A10),anti-Met(1:1000,Millipore,07-283),anti-Bcl2(1:500,CellSignaling,#2876S),anti-E2f3(1:500,Millipore,PG37),anti-Sirt1(1:1000,CellSignaling#2028),anti-cMyc(1:1000,CellSignaling,D84C12),andanti-a-Tubulin(Sigma,DM1A).
Theanti-p53antibody(1:300)wasakindgiftofKristianHelin(BRIC,Denmark).
SecondaryantibodieswereobtainedfromCellSignaling.
ECLreagentswereobtainedfromGEHealthcare.
WesternblotbandswerequantifiedusingImageJ.
ImmunohistochemistryMicewereirradiatedwith10Gyandsacrificed6hoursafter.
PFA-fixed,paraffin-embeddedsectionsweredeparaffinizedinxylene,andrehydrated.
ThesampleswerestainedwithCleavedCaspase-3antibody(CellSignaling,#9664)overnight,accordingtoCellSignalingprotocol.
Thesampleswerealsocounterstainedwith0.
1%alcoholicEosinYsolution(Sigma-Aldrich)or30%hematoxylin.
ThesectionswerethendehydratedandmountedinPermount(FisherScientific).
SamplepictureswerequantifiedusingImageJ.
SupportingInformationFigureS1RelativemiR-34expressioninmousetissuesuponirradiation.
(A)MiR-34aandmiR-34cexpressionbyNorthernblottingunderbasalconditionsand18hoursafterirradiation(10Gy).
(B)MiR-34a(leftpanel)andmiR-34c(rightpanel)expressionbyqPCRunderbasalconditionsand24hafterirradiation(10Gy).
Expressionlevelsoftreatedsampleswerenormalizedtountreatedsamples.
Errorbarsarestandarddeviations.
(PDF)FigureS2MacroscopiccharacterizationofmiR-34TKO/TKOmice.
(A)Representativepicturesofinternalorgansobtainedfromage-andsex-matchedwild-typeandmiR-34-nulladultmice.
(B)Scatterdotplotsshowingtotalbodyweightofage-matchedandsex-matchedwildtype(WT)andmiR-34-null(TKO)miceatdifferentages(upper-leftpanel)andrelativeweightofinternalorgans(remainingpanels;n$6pergenotype).
Errorbarsindicatemean+/21S.
E.
M.
P-valueswereobtainedusingtheunpairedtwo-tailedt-test.
(PDF)FigureS3MicroscopiccharacterizationofmiR-34TKO/TKOmice.
Representativeimagesofhematoxylinandeosinstainingofheart,kidney,liver,lung,smallintestine,ovary,testis,andspleen(blackscalebar,200mm),brain(greenscalebar,2000mm),andcolon(redscalebar,100mm)fromwild-typeandmiR-34TKO/TKOmice.
(PDF)FigureS4Completebloodcellcountofage-andsex-matchedwild-typeandmiR-34-deficientmice.
Peripheralbloodsamplesobtainedfromsex-andage-matchedadult(agerange3–16months)wild-type(WT)andmiR-34-null(TKO)miceweresubjectedtocompletebloodcellcount(n$5pergenotype).
Errorbarsindicatemean+/21S.
E.
M.
TheP-valueforeachparameterwascalculatedusingthetwo-tailedunpairedt-test.
Abbreviationsused:WBC=Whitebloodcells;RBC=redbloodcells;HCT=Hema-tocrit;HGB=Hemoglobin;MCV=MeanCorpuscolarVolume;MCH=MeanCorpuscolarHemoglobin;MCHC=MeanCorpus-colarHemoglobinConcentration.
(PDF)miR-34-NullMiceandp53PLoSGenetics|www.
plosgenetics.
org10July2012|Volume8|Issue7|e1002797FigureS5Serumchemistryofage-andsex-matchedwild-typeandmiR-34-deficientmice.
Samplesobtainedfromsex-andage-matchedadult(agerange3–16months)wild-typeandmiR-34TKO/TKOmiceweresubjectedtoastandardpanelofserumchemistryteststodetermineliverandkidneyfunction(n$5pergenotype).
Errorbarsindicatemean+/21S.
E.
M.
TheP-valueforeachassaywascalculatedusingthetwo-tailedunpairedt-test.
Abbreviationsused:ALP=AlkalinePhosphatase;ALT(SGPT),AlanineTransaminase;AST=Aspartatetransaminase;A/GRatio=(Albumin/GlobulinRatio).
(PDF)FigureS6Bonemarrow,spleenandthymusanalysisofage-andsex-matchedwild-typeandmiR-34TKO/TKOmice.
(A)Represen-tativecontourplotsshowinglymphoidandmyeloidcellpopula-tionsinthebonemarrow,spleen,andthymusofage-matchedandsex-matchedwild-type(n=6)andmiR-34TKO/TKOmice(n=6).
(B)Scatterdotplotssummarizingtheresultsoftheanalysesshownin(A).
Errorbarsare+/21standarddeviation.
P-valueswerecalculatedusingthetwo-tailedt-test.
(PDF)FigureS7Overallsurvivalofwild-typeandmiR-34TKO/TKOcohorts.
(A)Survivalcurvesforwild-typeandmiR-34TKO/TKOmice.
Agerangeofthecohortsis359–521days(mean:464days)forwild-typeand359–521days(mean:445days)formiR-34TKO/TKO.
P-value=1(log-ranktest).
(B)Survivalcurvesformousecohortswithindicatedgenotypesirradiatedwith1Gy2daysafterbirth.
Agerangeofthecohortsis298–425days(mean:333days)forwild-typeand387–425days(mean:401days)formiR-34TKO/TKO.
P-value=1(log-ranktest).
(PDF)FigureS8ExpressionofmiR-449a,miR-449bandmiR-449c.
(A)NorthernblotdetectionofmiR-449a,miR-449b,miR-449candmiR-34ainapaneloftissuesfromwildtypeandmiR-34TKO/TKOmice.
Foreachtissue,thesamemembranewasseriallyprobedfirstforthethreemembersofthemiR-449familyandlastlyformiR-34a.
RNAsfrommiR-34TKO/TKOtissueswereincludedtocontrolforcross-hybridization.
NoticethelossofsignalformiR-449binthemiR-34TKO/TKOlungandtestissamples,whichlikelyreflectscross-hybridizationofthemiR-449bprobetomiR-34.
(B–D)qPCRdetectionofmiR-449familymembersinMEFs(B),thymus(C),andspleen(D)ofwild-type,p532/2andmiR-34TKO/TKOmiceexposedtoDNAdamagingagents.
MEFsweretreatedwith0.
2mg/mldoxorubicinfor12hours.
Micewereirradiatedwith10Gyandeuthanized6hourslater.
NoticethattheallthreemembersofthemiR-449familyaredetectedatsignificantlylowerlevelscomparedtomiR-34,consistentwiththeNorthernblotanalysisshowninpanelA.
(PDF)TextS1Supportinginformationtexts.
Genotypingprotocolsandsupplementarymethodsareprovided.
(PDF)AcknowledgmentsWethankthemembersoftheVenturalaboratoryandJenniferHollensteinfordiscussingandeditingthemanuscript.
AuthorContributionsConceivedanddesignedtheexperiments:AVCPC.
Performedtheexperiments:AVCPCEYADPMCBPOY-CHJAVWPM.
Analyzedthedata:AVCPCY-CHEYADPMJAVCBWPM.
Wrotethepaper:AVCPC.
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miR-34-NullMiceandp53PLoSGenetics|www.
plosgenetics.
org12July2012|Volume8|Issue7|e1002797