MicroRNA125b expands hematopoietic stem cells and enriches for the lymphoidbalanced and lymphoidb.docx
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MicroRNA125b expands hematopoietic stem cells and enriches for the lymphoidbalanced and lymphoidb.docx
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MicroRNA125bexpandshematopoieticstemcellsandenrichesforthelymphoidbalancedandlymphoidb
MicroRNA-125bexpandshematopoieticstemcellsandenrichesforthelymphoid-balancedandlymphoid-biasedsubsets
1.A.G.LisaOoia,
2.DebashisSahooa,
3.MaddalenaAdornoa,
4.YuleiWangb,
5.IrvingL.Weissmana,1,and
6.ChristopherY.Parka,1,2
+AuthorAffiliations
1.aInstituteofStemCellBiologyandRegenerativeMedicineandDepartmentsofPathology,ChemicalandSystemsBiology,andDevelopmentalBiology,StanfordUniversitySchoolofMedicine,Stanford,CA94305;and
2.bAppliedBiosystems,Inc.,FosterCity,CA94404
∙↵2Presentaddress:
MemorialSloan-KetteringCancerCenter,1275YorkAvenue,NewYork,NY10065.
1.ContributedbyIrvingL.Weissman,November3,2010(sentforreviewSeptember25,2010)
NextSection
Abstract
MicroRNAsprofoundlyimpacthematopoieticcellsbyregulatingprogenitorcell-fatedecisions,aswellasmatureimmuneeffectorfunction.Howevertodate,microRNAsthatregulatehematopoieticstemcell(HSC)functionhavebeenlesswellcharacterized.HereweshowthatmicroRNA-125b(miR-125b)ishighlyexpressedinHSCsanditsexpressiondecreasesincommittedprogenitors.OverexpressionofmiR-125binmouseHSCenhancestheirfunction,demonstratedthroughserialtransplantationofhighlypurifiedHSC,andenrichesforthepreviouslydescribedSlamf1loCD34−lymphoid-balancedandtheSlamf1negCD34−lymphoid-biasedcellsubsetswithinthemultipotentHSC(CD34-KLS)fraction.MatureperipheralbloodcellsderivedfromthemiR-125b–overexpressingHSCareskewedtowardthelymphoidlineage.Consistentwiththisobservation,miR-125boverexpressionsignificantlyincreasesthenumberofearlyB-progenitorcellswithinthespleenandinducestheexpansionandenrichmentofthelymphoid-balancedandlymphoid-biasedHSCsubsetviaanantiapoptoticmechanism,reducingthemRNAexpressionlevelsoftwoproapoptotictargets,BmfandKLF13.TheantiapoptoticeffectofmiR-125bismorepronouncedinthelymphoid-biasedHSCsubsetbecauseoftheirintrinsichigherbaselinelevelsofapoptosis.TheseeffectsofmiR-125bareassociatedwiththedevelopmentoflymphoproliferativedisease,markedbyexpansionofCD8+Tlymphocytes.Takentogether,thesedatarevealthatmiR-125bregulatesHSCsurvivalandcanpromotelymphoid-fatedecisionsattheleveloftheHSCbypreferentiallyexpandinglymphoid-balancedandlymphoid-biasedHSC.
MicroRNAsareaclassofevolutionarilyconservedsmallRNAsthatinducecleavageortranslationalrepressionoftargetmRNAsbybindingtopartiallycomplementaryseedsequencesfoundonthe3′UTRregionsoftargetmRNAs
(1).MicroRNAsarepredictedtoprofoundlyaffectgene-expressionprofilesandregulatetheexpressionofhundredsofmRNAs(1–3).
SeveralstudieshavedemonstratedthatmiRNAsmayregulatelineage-fatedecisionsinhematopoieticdevelopment.Forexample,ectopicexpressionofmiR-181inlineage-negativemousebone-marrowcellsleadstoexpansionofBcellsbutadiminutionofTcells(4).Myeloid-specificmiR-223hasbeenimplicatedingranulocyticdevelopment,withmiR-223knockoutmiceexhibitingincreasednumbersofgranulocyteprogenitorsandmaturecells(5).DeficiencyofmiR-150leadstoexpansionoftheB1B-cellcompartment,whereasectopicexpressionofmiR-150impairsBcelldevelopment(6).Inaddition,miR-150,whichishighlyexpressedinthemegakaryocyticlineage,canbiasdifferentiationofmegakaryocyte-erythroidprogenitors(MEP)towardthemegakaryocyticfateattheexpenseoferythrocytes(7).Morerecently,studiessuggestthatmiRNAs,suchasmiR-29a,mayregulatehematopoieticstemcell(HSC)self-renewal,asevidencedbytheaberrantinductionofself-renewalinprogenitorpopulationsbymiRNAshighlyexpressedinHSCandhumanacutemyeloidleukemia(AML)(8).
WeareinterestedinidentifyinggenesthatregulateHSCfunctionandundertookanefforttoidentifymiRNAsthataredifferentiallyexpressedinHSC.WefoundthatmiR-125bisexpressedathighestlevelsinmouseHSC,andthatmiR-125bexpressiondecreasesprogressivelyascellsdifferentiatetomyeloidandlymphoidcommittedprogenitors,withmiR-125bexpressedatsignificantlyhigherlevelsincommonlymphoidprogenitors(CLP)comparedwiththecommonmyeloidprogenitors(CMP).TotestthebiologicalfunctionofmiR-125binHSC,weoverexpressedmiR-125binhighlypurifiedHSCusinglentiviralvectors.OverexpressionofmiR-125bincreasedHSCengraftmentincompetitivetransplants,andweconfirmedthatthiseffectwasaresultofcell-autonomouseffectsonHSCandnotcommittedprogenitorsbyrecapitulatingthephenotypethroughserialtransplantationofhighlypurifiedHSC.Inaddition,miR-125binducedanexpansionoftheHSCcompartmentinpartbyinhibitingexpressionofatleasttwoantiapoptotictargetgenes,Bmf(Bcl2modifyingfactor)andKLF13(Krueppel-likefactor13).BothtargetswereidentifiedaspotentialmiR-125btargetsinvivobyevaluatingpurifiedstemandprogenitorpopulations.TheHSCexpansionwasassociatedwithalymphoiddifferentiationbiasintheperipheralblood.InasmallfractionofthemiR-125btransplantedmice,weobservedalymphoproliferativedisease.
Concurrently,O'Connelletal.(9)foundthatmiR-125bishighlyexpressedinthestemandprogenitor-cellcompartmentofthemousebonemarrowandthat1,000-foldoverexpressionofmiR-125binthehematopoieticstemandprogenitorpopulationsgaverisetoamyeloproliferativediseasethatprogressedtoAML.Guoetal.(10)alsofoundthatmiR-125aishighlyexpressedinthestem-andprogenitor-cellcompartmentofthebonemarrow.Overexpressionofmir-125aalsoexpandedtheHSCcompartmentviaanantiapoptoticmechanism,possiblybytargetingtheproapoptoticprotein,Bak1.OurobservationsconfirmthesefindingsandextendthembydemonstratingthatmiR-125bcaninducepreferentialexpansionofthepreviouslydescribedlymphoid-balancedandlymphoid-biasedHSC(phenotypicSlamloCD34−andSlamnegCD34−KLS)populations.
PreviousSectionNextSection
Results
ExpressionProfilingofHematopoieticPopulations.
ToidentifymiRNAsthatmayregulateHSCfunction,wefirstanalyzedthemiRNAexpressionprofilesofmultiplehematopoieticpopulations.Wedouble-sortedHSCandcommittedprogenitorpopulationsfromthebonemarrowofC57BL/6-Thy1.1(BA)micebasedoncell-surfacemarkersdefinedbyourlaboratoryandothers(11):
HSC(c-kit+Sca-1+Lin−Flk2−CD34−),multipotentprogenitor(MPP)flk−cells(c-kit+Sca-1+Lin−Flk2−CD34+),MPPflk+cells(c-kit+Sca-1+Lin−Flk2+CD34+),CLPcells(Ly6c−Flk2+IL7R+CD27+CD4−B220−CD19−CD11c−),CMPcells(c-kit+Sca-1−Lin−FcGloCD34+),granulocyte-monocyteprogenitor(GMP)cells(c-kit+Sca-1−Lin−FcGhiCD34+),andMEPcells(c-kit+Sca1−Lin−FcGloCD34−).MicroRNAsweremeasuredfromtotalRNAisolatedfromeachcellpopulationusingapreviouslydescribedTaqManreal-timePCRstrategy(8).Fiveindependentlysortedbiologicreplicates,eachrepresentingfivepooledmice,wereusedforthesestudies.OuranalysisrevealedthatmiR-125bexpressionisconsistentlyandsignificantlyhigherinHSC(>twofold,P<0.05)comparedwithallotherprogenitorpopulationsassayed(Fig.1).Inparticular,theexpressionlevelofmiR-125bissignificantlyhigherintheCLPpopulationcomparedwiththeCMPpopulation.AsimilartrendwasobservedinhumanHSCandcommittedprogenitorcellpopulations;however,thedifferenceswerenotstatisticallysignificant(P>0.05comparingHSCwithallprogenitorpopulations),likelyreflectingthegeneticheterogeneityofthehumancellpopulations.
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Fig.1.
MicroRNA-125bishighlyexpressedinHSCsandMPPcellsinmiceandhumans.(A)NormalizedexpressionlevelofmiR-125bwasdeterminedbyquantitativePCRusingmiRNATaqmanprobesindouble-sortedmousehematopoieticcellpopulations:
HSC(c-kit+Sca1+Lin−CD34−Flk2−),MPPFlk−(c-kit+Sca1+Lin−CD34+Flk2−),MPPFlk+(c-kit+Sca1+Lin−CD34+Flk2+),CLP(Ly6c−Flk2+IL7R+CD27+CD4−B220−CD19−CD11c−),CMP(c-kit+Sca-1−Lin−FcGloCD34+),GMP(c-kit+Sca-1−Lin−FcGhiCD34+),andMEP(c-kit+Sca1−Lin−FcGloCD34−BM).Expressionwasnormalizedagainstmmu-mir-16(n=5).(B)NormalizedexpressionlevelsofmiR-125bweredeterminedbyquantitativePCRusingmiRNATaqmanprobesindouble-sortedbonemarrowhumanhematopoieticcellpopulations:
HSC(Lin−CD34+CD38−CD90+CD45RA−),MPP(Lin−CD34+CD38−CD90−CD45RA−),CMP(Lin−CD34+CD38+CD123+CD45RA−),GMP(Lin−CD34+CD38+CD123+CD45RA+),andMEP(Lin−CD34+CD38+CD123loCD45RA−).Expressionwasnormalizedagainstsno-R2.(n=5).Thedifferenceswerenotstatisticallysignificant,possiblybecauseofheterogeneityofthehumanhematopoieticpopulations.ErrorbarsdenoteSEM.
Mir-125bexistsintwolocationsinthemouseandhumangenomes.Mmu-miR-125b1,foundonmousechromosome9,andhsa-miR-125b1,foundonhumanchromosome11,existinaputativepolycistronicclusterwithmiR-let7a2andmiR-100(Fig.S1).Mmu-miR-125b2(mousechromosome16)andhsa-miR-125b2(humanchromosome21)existinaclusterwithmiR-let7c1andmiR-99a.Interestingly,notallmiRNA-125bclustermembershaveexpressionlevelsthatcovarywithmiR-125binearlyhematopoieticprogenitors.Specifically,onlymiR-99aandmiR-100appeartodemonstratesimilarprofilestomiR-125b,withhighestexpressionlevelsinHSCandgraduallydecreasinglevelsastheprogenitorsdifferentiate.BothmiR-99aandmiR-100alsodemonstratehigherexpressionlevelsintheearlylymphoidprogenitorscomparedwiththeearlymyeloidprogenitors.TheseresultssuggestthatthesemiRNApolycistronsmayberegulatedbyposttranscriptionalmechanismsinearlyhematopoiesis.
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