ofSeptember21,2017.
Thisinformationiscurrentas
CellsviaTargetingRunx1
andFunctionofMyeloid-DerivedSuppressor
MicroRNA-9RegulatestheDifferentiation
Wang
XinyuTian,YueZhang,HuaxiXu,LiweiLuandShengjun
JieTian,KeRui,XinyiTang,JieMa,YungangWang,
http://www.jimmunol.org/content/195/3/1301
doi:10.4049/jimmunol.1500209
June2015;
2015;195:1301-1311;Prepublishedonline19JImmunol
Material
Supplementary
9.DCSupplemental
http://www.jimmunol.org/content/suppl/2015/06/19/jimmunol.150020
References
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Copyright?2015byTheAmericanAssociationof
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TheJournalofImmunology
MicroRNA-9RegulatestheDifferentiationandFunctionof
Myeloid-DerivedSuppressorCellsviaTargetingRunx1
JieTian,
,?,1
KeRui,
?,1
XinyiTang,
?
JieMa,
?
YungangWang,
?
XinyuTian,
?
YueZhang,HuaxiXu,
?
LiweiLu,
?
andShengjunWang
,?
Myeloid-derivedsuppressorcells(MDSCs)playacriticalroleintumor-associatedimmunosuppression,thusaffectingeffective
immunotherapiesforcancers.However,themolecularmechanismsinvolvedinregulatingthedifferentiationandfunctionof
MDSCsremainlargelyunclear.Inthisstudy,wefoundthatinhibitionofmicroRNA(miR)-9promotedthedifferentiationofMDSCs
withsignificantlyreducedimmunosuppressivefunctionwhereasoverexpressionofmiR-9markedlyenhancedthefunctionof
MDSCs.Notably,knockdownofmiR-9significantlyimpairedtheactivityofMDSCsandinhibitedthetumorgrowthofLewislung
carcinomainmice.Moreover,miR-9regulatedMDSCsdifferentiationbytargetingtherunt-relatedtranscriptionfactor1,anes-
sentialtranscriptionfactorinregulatingMDSCdifferentiationandfunction.Furthermore,theCREBwasfoundtoregulatemiR-9
expressioninMDSCs.Takentogether,ourfindingshaveidentifiedacriticalroleofmiR-9inregulatingthedifferentiationand
functionofMDSCs.TheJournalofImmunology,2015,195:1301–1311.
M
yeloid-derivedsuppressorcells(MDSCs)areahet-
erogeneouspopulationofimmaturemyeloidcells,
includingmyeloidprogenitors,precursorsofmacro-
phages,dendriticcellsandgranulocytes,whicharecharacterized
bytheirstrongabilitytosuppressTcellfunctions(1,2).Inmice,
MDSCsarebroadlydefinedascellscoexpressingtwophenotypic
markersCD11bandGr-1(1).MurineMDSCscanbefurther
subdividedintotwodifferentsubsetsbasedontheirexpressionof
Ly-6CandLy-6G(3,4).CD11b
+
Ly-6G
+
Ly-6C
low
cellsshowing
granulocyte-likemorphologyaretermedgranulocyticMDSCs
(G-MDSCs),whereasCD11b
+
Ly-6G
2
Ly-6C
high
cellswith
monocytic-likemorphologyaretermedmonocyticMDSCs
(M-MDSCs).UnlikemurineMDSCs,thehumanMDSCsareless
welldefinedowingtothelackofspecificmarkers.MDSCsare
commonlydefinedasCD11b
+
CD33
+
HLA-DR
low/2
cells.MDSCs
inhumansarealsosubdividedintotwomainsubsets:CD15
+
CD14
2
CD11b
+
CD33
+
HLA-DR
low/2
G-MDSCsandCD15
2
CD14
+
CD11b
+
CD33
+
HLA-DR
low/2
M-MDSCs(5–7).Differentsubsets
ofMDSCsusedifferentialpathwaystosuppressTcellfunction.
G-MDSCssuppressTcellproliferationmainlybyexpressinghigh
levelsofarginase1(ARG1)andreactiveoxygenspecies(ROS)
whereasM-MDSCsexpressinducibleNOsynthase(iNOS)in
additiontohighlevelsofARG1(1,4,8,9).Inhealthyindividuals,
MDSCsaregeneratedinbonemarrowandquicklydifferentiate
intomaturemacrophages,dendriticcells,orgranulocytes.How-
ever,apartialblockinthedifferentiationofMDSCsintomature
myeloidcellsoftenoccursunderpathologicalconditions,resulting
intheexpansionofthispopulation(1).BecauseMDSCsare
criticallyinvolvedintumor-associatedimmunesuppression,
manystudieshavefocusedonexploringtherapeuticstrategies
toeliminatethesecellsortomodulatetheirfunction.Astheim-
munosuppressiveactivityofMDSCsismostlyassociatedwith
theirimmaturestate,furtherstudiesonthemodulationofMDSCs
differentiationwillbeofgreatinterestinreversingtheirimmu-
nosuppressivefunctionswithinthetumormicroenvironment.
b-Glucanshavebeenrecognizedasbiologicalresponsemodi-
fiers,whichcanenhancebothinnateandadaptiveimmune
responses(10,11).Dectin-1,anon–Toll-likepatternrecognition
receptorforb-glucan,ispredominantlyexpressedonmyeloid
cells,includingdendriticcells,monocytes/macrophages,neu-
trophils,andasubsetofTcells(12,13).Ourpreviousstudieshave
shownthatbothM-MDSCsandG-MDSCsexpressdectin-1(14).
Moreover,wholeb-glucanparticles(WGPs),theparticulate
b-glucanderivedfromyeastSaccharomycescerevisiae,could
promoteM-MDSCdifferentiationintomorematuremyeloidcells
viathedectin-1pathway,thuseffectivelyabrogatingM-MDSC–
mediatedimmunesuppressionandenhancingantitumorresponses
(14).However,themolecularmechanismsunderlyingthefunc-
tionalmodulationofMDSCsremainlargelyunclear.
ThemicroRNAs(miRNAs)areshortandsingle-strandednon-
codingRNAs(20–23nucleotides)thatplaycrucialrolesinregulating
geneexpressionattheposttranscriptionallevel.Itbecomesclearthat
miRNAsbindtothespecificcognatesequencesinthe39untrans-
latedregion(UTR)oftargetmRNAstomediateposttranscriptional
generepressionbydegradingtargettranscriptsorinhibitingprotein
DepartmentofLaboratoryMedicine,AffiliatedPeople’sHospital,JiangsuUniver-
sity,Zhenjiang212002,China;
?
InstituteofLaboratoryMedicine,JiangsuKeyLab-
oratoryofLaboratoryMedicine,JiangsuUniversity,Zhenjiang210013,China;and
?
DepartmentofPathology,UniversityofHongKong,HongKong999077,China
1
J.T.andK.R.contributedequallytothiswork.
ReceivedforpublicationJanuary30,2015.AcceptedforpublicationMay21,2015.
ThisworkwassupportedbyNationalNaturalScienceFoundationofChinaGrants
31170849and31470881,SpecializedProjectforClinicalMedicineofJiangsuProv-
inceGrantBL2014065,SpecializedResearchFundfortheDoctoralProgramof
HigherSchoolGrant20133227110008,HealthDepartmentFoundationofJiangsu
ProvinceGrantZ201312,andbyJiangsuProvince“333”Project,PriorityAcademic
ProgramDevelopmentofJiangsuHigherEducationInstitutions.
ThemicroarraydatapresentedinthisarticlehavebeensubmittedtotheGene
ExpressionOmnibus(http://www.ncbi.nlm.nih.gov/geo/)underaccessionnumber
GSE67578.
AddresscorrespondenceandreprintrequeststoProf.ShengjunWang,Departmentof
LaboratoryMedicine,AffiliatedPeople’sHospital,JiangsuUniversity,Zhenjiang
212002,China.E-mailaddress:sjwjs@ujs.edu.cn
Theonlineversionofthisarticlecontainssupplementalmaterial.
Abbreviationsusedinthisarticle:ARG1,arginase1;G-MDSC,granulocytic
MDSC;iNOS,inducibleNOsynthase;LLC,Lewislungcarcinoma;MDSC,
myeloid-derivedsuppressorcell;miR,microRNA;miRNA,microRNA;M-MDSC,
monocyticMDSC;Mut,mutation-type;qRT-PCR,quantitativereal-timePCR;
ROS,reactiveoxygenspecies;Runx1,runt-relatedtranscriptionfactor1;siRNA,
smallinterferingRNA;UTR,untranslatedregion;WGP,wholeb-glucanparticle;
WT,wild-type.
CopyrightC2112015byTheAmericanAssociationofImmunologists,Inc.0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500209
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translation(15).miRNAshavebeenshowntoregulatethe
differentiation,maturation,andfunctionofimmunecells.
Moreover,miRNAsplayanessentialroleinthemaintenanceof
thecellularhomeostasisandthedevelopmentofvariousphysi-
ologicalsystems(16–20).ManymiRNAsidentifiedfrommouse
bonemarrowcellsaredifferentiallyregulatedinvarioushema-
topoieticlineages(21).However,itisstillnotclearwhetherand
howmiRNAsareinvolvedinregulatingMDSCdifferentiation
andfunction.
Inthisstudy,wefoundthatmicroRNA(miR)-9played
acriticalroleinregulatingthedifferentiationandfunctionof
MDSCs.TreatmentwithWGPsmarkedlydownregulatedmiR-9
expressioninmouseMDSCs,aneffectspecificallyabolished
bysupplementingmiR-9.Moreover,downregulationofmiR-9
canpromotethedifferentiationofMDSCsanddecreasetheir
suppressivecapacity.Furthermoreweidentifiedrunt-related
transcriptionfactor1(Runx1),aregulatorindifferentiationand
functionofMDSCs,asthedirecttargetofmiR-9.Thus,these
findingsprovidenewinsightsinunderstandingthemolecular
mechanismsthatregulatethedifferentiationandfunctionof
MDSCs.
MaterialsandMethods
Cellline,mice,andtumormodels
TheLewislungcarcinoma(LLC)cells,humanembryonickidneycellline
293Tcellsandmousemonocyte/macrophagecelllineRAW264.7were
purchasedfromtheAmericanTypeCultureCollection.MaleC57BL/6
micewerehousedinaspecificpathogen-freeanimalfacilityandused
at6–8wkofage.Fortumormodels,micewereimplanteds.c.with
LLC(3310
6
/mouse)(14).
IsolationofMDSCs
MurineMDSCswereisolatedfromthespleensofLLCtumor-bearing
miceusingamouseMDSCisolationkit(MiltenyiBiotec)followingthe
manufacturer’sprotocol.ThepurityofCD11b
+
Ly-6G
+
Ly-6C
low
G-MDSCs
was.98%andthepurityofCD11b
+
Ly-6G
2
Ly-6C
high
M-MDSCswas
.90%.TotalMDSCswereisolatedusinganti-CD11bconjugatedtobiotin
followedbyanti-biotinmicrobeads(MiltenyiBiotec),andthepurityof
CD11b
+
Gr-1
+
cellswas.98%.ForMDSCstimulation,b-glucanWGP
(Biothera)wasprovidedbyDr.JunYanfromtheUniversityofLouisville
SchoolofMedicine(22).
Flowcytometry
Forsurfacemarkers,single-cellsuspensionswerestainedwithrelevant
fluorochrome-conjugatedCD11c,F4/80,CD40,CD80,CD86,andMHC
classIImAbs(eBioscience).CellswerestainedwithaboveAbsinPBSfor
FIGURE1.Runx1isinvolvedinthedifferentiationandfunctionofMDSCs.LLCcells(3310
6
)wereinjecteds.c.intoC57BL/6mice.After4wk,
splenocyteswerecollectedtoisolateM-MDSCsandG-MDSCs.(A)M-MDSCsandG-MDSCswereculturedinthepresenceorabsenceofWGPs(100mg/ml)
for48h;alowlevelofGM-CSF(1ng/ml)wassupplementedtokeepcellviability.Then,freshlyisolatedMDSCsandMDSCstreatedwithorwithoutWGPs
werelysed,andRunx1levelswereanalyzedbyWesternblottinganalysis.b-Actinservedasaloadingcontrol.(B–E)M-MDSCsorG-MDSCstransfectedwith
Runx1siRNA(siRunx1)ornegativecontrolwerestimulatedwithWGPsfor48h.(B)qRT-PCRwasusedtoanalyzetheinterferenceefficiency.(C)
MDSCsweretreatedwithWGPsandthenwerestainedwithspecificAbsagainstCD11c,F4/80,CD40,CD80,CD86,andMHCclassII.(DandE)
ArginaseactivityandROSlevelsweremeasuredasdescribedinMaterialsandMethods,andqRT-PCRwasperformedforiNOSmRNAexpression.
(FandG)M-MDSCs(F)andG-MDSCs(G)weretransfectedwithRunx1siRNA(siRunx1)ornegativecontrol;after24h,cellswereharvestedand
thencoculturedwithrespondercells,CD4
+
Tcells(left),orCD8
+
Tcells(right)(MDSC/Tcellratioof2:1,1:1,0.5:1)inthepresenceofanti-CD3
mAbandanti-CD28mAbfor72h.SuppressivityonTcellproliferationwasmeasuredbyincorporationof[
3
H]thymidine.Statisticalsignificanceis
evaluatedbetweencontrolgroupandsiRunx1groupatdifferentratios.Alldataareshownasmeans6SDpooledfromthreeindependent
experiments.p,0.05,p,0.01,p,0.001.Ctrl,control.
1302miR-9REGULATESMDSCsBYTARGETINGRunx1
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30minat4?C.FlowcytometrywasperformedusingFACSCaliburflow
cytometer(BectonDickinson).
RNAisolationandquantitativereal-timePCR
TotalRNAofcellswasextractedinTRIzol(Invitrogen).Then,cDNAwas
synthesizedwithReverTraAceqPCRRTkit(Toyobo)accordingtothe
manufacturer’sinstructions.TheRT-PCRandquantitativereal-timePCR
(qRT-PCR)wereperformedaspreviouslydescribed(23).b-Actinwas
usedasaninternalcontrol.TheprimersarelistedinSupplementalTableI.
ForqRT-PCRanalysesofmiRNAusingSYBRRT-PCRkit(TakaraBio),
sequencesofRTprimerswithstem-loopstructureare:U6,59-CGCTTCAC-
GAATTTGCGTGTCAT-39;mmu-miR-9,GTCGTATCCAGTGCGTGTCGTG-
GAGTCGGCAATTGCACTGGATACGACTCATACA-39;mmu-miR-181d,59-
GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGA-
CACCCAC-39;mmu-miR-181b,59-GTCGTATCCAGTGCGTGTCGTG-
GAGTCGGCAATTGCACTGGATACGACACCCACCG-39;hsa-miR-9,
59-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTG-
GATACGACTCATACA-39.qRT-PCRprimersarelistedinSupplemental
TableI.TherelativeexpressionofmiRNAswasnormalizedtothatof
internalcontrolU6.RelativequantificationofmRNAexpressionwas
calculatedbythecomparativethresholdcyclemethod.
Microarrayanalysis
ThemiRNAmicroarrayassaywasconductedatKangChenBio-tech.Total
RNAwasextractedusingTRIzolandanmiRNeasyminikit(Qiagen)
accordingtothemanufacturer’sinstructions.AfterpassingRNAquantity
measurementbytheNanoDrop1000,sampleswerelabeledusingthe
miRCURYHy3/Hy5powerlabelingkitandhybridizedonthemiRCURY
LNAarray(v.16.0).Followingthewashingsteps,theslideswerescanned
usingtheAxonGenePix4000Bmicroarrayscanner.Scannedimageswere
thenimportedintoGenePixPro6.0software(Axon)forgridalignment
anddataextraction.ReplicatedmiRNAswereaveragedandmiRNAsthat
hadintensities.50inallsampleswerechosenforcalculatingnormali-
zationfactor.Expresseddatawerenormalizedusingmediannormalization.
Afternormalization,differentiallyexpressedmiRNAswereidentified
throughfoldchangefiltering.Thethresholdusedtoscreenup-ordown-
regulatedmiRNAsisfoldchange$2.0.Themicroarraydatahavebeen
submittedtotheGeneExpressionOmnibus(http://www.ncbi.nlm.nih.gov/
geo/query/acc.cgi?acc=GSE67578)underaccessionnumberGSE67578.
Westernblotanalysis
Proteinsextractedfromcellswerepreparedaspreviouslydescribed(23).
ProteinsampleswereseparatedbySDS-PAGE,thentransferredonto
Immobilonpolyvinylidenedifluoridemembranes(Bio-Rad)andprobed
withrabbitRunx1Ab(CellSignalingTechnology)andmouseb-actinAb
(Abcam)followedbychemiluminescentdetection(ChampionChemical).
DetectionofarginaseactivityandROSlevels
Arginaseactivity,determiningtheconversionofargininetoornithineand
urea,wasdetectedbyaquantitativecolorimetricassayemployinga
QuantiChromarginaseassaykit(BioAssaySystems).Thearginaseac-
tivitywascalculatedaccordingtothemanufacturer’sinstructions.
Theoxidation-sensitivedye29,79-dichlorofluoresceindiacetate(Invi-
trogen)wasusedtodetectROSproductionbyMDSCs.Cellsweresimul-
taneouslyculturedwith2.5mM29,79-dichlorofluoresceindiacetatewith
30ng/mlPMAinPBSfor30minbeforeflowcytometricanalysis.
MDSCsuppressionassay
ForevaluationofMDSCsuppressivefunction,MDSCsisolatedfrom
spleensoftumor-bearingmiceweretransfectedwithmiR-9mimics,miR-9
inhibitors,andRunx1smallinterferingRNA(siRNA)oranegativecontrol
formiRNAmimics,inhibitor,andsiRNA(RiboBio).Forrespondercells,
splenicCD4
+
TcellsandCD8
+
Tcellsweresortedfromwild-type(WT)
C57BL/6miceusingCD4microbeads(MiltenyiBiotec),FITC-conjugated
anti-CD8mAb(BDPharmingen),andanti-FITCmicrobeads(Miltenyi
Biotec).ThepuritiesofCD4
+
TcellsandCD8
+
Tcellswere.95%.
Respondercellswerecoculturedwithdifferentratiosoftransfected
MDSCs(withoutgammairradiation)inU-bottom96-wellplates(Costar)in
thepresenceofanti-CD3(BioLegend,10mg/ml)andanti-CD28(Bio-
Legend,5mg/ml)mAbsfor72handpulsedwith[
3
H]thymidine(Pharmacia
Biotech,1mCi/well)forthelast16hofculture.Thecapacityof
MDSCstosuppressTcellswascalculatedbycpmMDSCsplusTcells2cpm
MDSCs,andtheaveragevaluesofMDSCcpmunderdifferenttreat-
mentwere,120.
Transfection
miR-9mimics,miR-9inhibitors,miR-9agomir,miR-9antagomir,Runx1
siRNA,andnegativecontrolsweresynthesizedbyRiboBio.Oligonucle-
otidetransfectionwasperformedwithEntranster-R(EngreenBiosystem)
accordingtothemanufacturer’sinstructions.
39UTRluciferasereporterassays
TheWTRunx139UTRluciferasereportervectorswereconstructedby
amplifyingthemouseRunx139UTRandclonedintotheXhoIsiteofthe
psiCHECK-2vector(Promega).ThemutationtypeofRunx139UTRwas
obtainedfromtheWTconstructbyoverlap-extensionPCR.293Tcells
werecotransfectedwith100ngluciferasereporterplasmidand100nM
miR-9mimicsusingLipofectamine2000reagents(Invitrogen)according
FIGURE2.DownregulationofmiR-9
inMDSCsstimulatedbyWGPs.(A)
SortedsplenicMDSCsfromtumor-bear-
ingmicewerestimulatedwith100mg/ml
WGPsfor24h,andtheexpressionof
miR-9,miR-181d,andmiR-181bwas
detectedbyqRT-PCRandnormalizedto
theexpressionofU6.(BandC)Isolated
M-MDSCsandG-MDSCsfromtumor-
bearingmicespleensweretreatedwith
100mg/mlWGPs,andqRT-PCRwas
usedtoanalyzethe(B)miR-181dand
(C)miR-9expression.(D)Thepurified
M-MDSCsandG-MDSCswerepre-
treatedwithanti–dectin-1mAborratIgG
(5mg/ml)for1hat37?Candthentreated
with100mg/mlWGPs;after24hstim-
ulation,miR-9expressionwasdetected
byqRT-PCR.Dataareshownasmeans6
SDofthreeindependentexperiments.p,
0.05,p,0.01,p,0.001.
TheJournalofImmunology1303
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tothemanufacturer’sinstructions.After24h,lysatesof293Tcellswere
harvestedusinglysisbuffer(Promega),andluciferaseactivitieswere
measuredusingDual-Luciferasereporterassaysystem(Promega)accord-
ingtothemanufacturer’sinstructions.Datawerenormalizedfortransfec-
tionefficiencybydividingfireflyluciferaseactivitywiththatofRenilla
luciferase.
Promoteranalysis
A2-kbfragmentlocatedupstreamofthemiR-9transcriptionstartsite
wasPCRamplifiedandinsertedintopGL3-Basicvector(Promega).The
mutationtypelackingCREBbindingsiteswasobtainedfromtheWT
constructbyoverlap-extensionPCR.TheWTplasmids(pGL3–miR-9
promoter)ormutation-typeplasmids(MutpGL3–miR-9promoter),con-
trolplasmid(pRL-TK)togetherwithCREBoverexpressionplasmids,or
controlplasmids(pcDNA3.1)werecotransfectedinto293Tcells.After
24h,cellswereassayedwiththeDual-Luciferaseassay(Promega).
InvivoMDSCsuppressiveexperiments
MDSCstransfectedwithmiR-9agomir,miR-9antagomir,ornegativecontrols
wereinjectedintothetumorsofmiceondays1and7(2310
6
/mouse)after
palpabletumorswereformed.TheliveMDSCstransfectedwithBLOCK-iT
fluorescentoligonucleotides(Invitrogen)canbemeasuredinvivoevenafter
injectionfor5d(SupplementalFig.1A–H).
Patientsandtissuesamples
Humanprimarytumortissuesanddistalnoncanceroustissueswere
collectedfrompatientsclinicallydiagnosedwithlungcancerat
theAffiliatedPeople’sHospitalofJiangsuUniversity(Zhenjiang,
China).Tissuesampleswereimmediatelysnapfrozeninliquidnitrogen.
Bothtumorsanddistalnoncanceroustissueswerehistologically
examined.
FIGURE3.miR-9suppressesRunx1expressionbydirectlytargetingits39UTR.(A)SchematicrepresentationofthepredictedRunx139UTRindicating
thebindingsitesformiR-9,anddesignedmutatedversionoftheRunx139UTR.(B)Constructedluciferasereportervectors.Luciferasereportervectors
wereconstructedinpsiCHECK-Runx139UTRwiththefollowing:1)seedsequence1(185–190,Runx139UTR1)andseedsequence2(653–658,Runx1
39UTR2),orRunx139UTR(1+2);2)mutatedseedsequence1andseedsequence2,orRunx139UTR1Mut+2);3)seedsequence1andmutatedsequence2,
orRunx139UTR2Mut+1);and4)mutatedseedsequence1andmutatedseedsequence2,orRunx139UTR(1+2)Mut.TheRunx139UTRwasclonedand
mutatedbyPCR-basedmutagenesisaccordingtotheprotocolinMaterialsandMethods.(C)293Tcells(1310
5
)werecotransfectedwith100nMmiR-9
mimics(miR-9)ormiRcontroltogetherwith200ngWTormutantRunx139UTR(1+2),orRunx139UTR1Mut+2,Runx139UTR2Mut+1orRunx1
39UTR(1+2)Mut,asindicated.After24h,thefireflyluciferaseactivitywasmeasuredandnormalizedbyRenillaluciferaseactivity.(DandE)RAW264.7
cellsweretransfectedwithdifferentamountsofmiR-9mimics(0,10,50,100nM)ormiRcontrolfor24h,andrelativeexpressionofmiR-9(D)andRunx1
(E)wasanalyzedbyqRT-PCR.(F)RAW264.7cellsweretransfectedwith100nMmiR-9mimics,Runx1siRNA(siRunx1),oranegativecontrolcontaining
miRNAmimicsandsiRNAnegativecontrololigonucleotides.After24h,WesternblottingwasusedtodetectRunx1proteinlevel.Quantitationofthe
Runx1/b-actinratioisshown.Dataareshownasmean6SDofthreeindependentexperiments.p,0.01,p,0.001.Ctrl,control.
1304miR-9REGULATESMDSCsBYTARGETINGRunx1
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Immunofluorescencemicroscopy
ParaffinsectionswereincubatedwithFITC-conjugatedanti-CD11bmAb
(eBioscience)andallophycocyanin-conjugatedanti-CD33mAb(eBio-
science).Matchedisotypecontrols(eBioscience)wereusedforcontrol
staining.AfterthreewashesinPBS,thesectionswereincubatedwith
Hoechst33342(Beyotime)forlabelingnuclei.
RNAextractionfromserumandqRT-PCRanalysisformiRNA
Bloodsamplesoflungcancerpatientsandhealthysubjectswerecollected
andthencentrifugedtoobtainserum.ANucleoSpinmiRNAplasmakit
(Macherey-Nagel)wasusedtoextractmiRNAinplasma.cDNAofthe
miRNAwassynthesizedwithmiRNA-specificRTprimersinthetran-
scriptionRTmixture(TakaraBio)(RTbuffer,dNTPmixture,Moloney
murineleukemiavirus,RNaseinhibitor),withtheprocedureincluding42?C
for60minand70?Cfor10min.qRT-PCRanalysesofmiRNAusedthe
SYBRRT-PCRkit(TakaraBio)andfollowedtherecommendedcycling
conditions(denaturationat95?Cfor30sfollowedby35cyclesof95?Cfor
5s,60?Cfor15s,and72?Cfor15s).TherelativeexpressionofmiRNAs
wasnormalizedtothatofinternalcontrolU6.Relativequantificationof
mRNAexpressionwascalculatedbythecomparativethresholdcycle
method.
Computationalprediction
Twotargetpredictiondatabases,TargetScan6.1(http://www.targetscan.
org)andmiRanda(http://www.microrna.org/),wereusedtoanalyzethe
interactionsbetweenmiR-9andRunx139UTR.PutativeCREBbinding
sitewithinthe2-kb59flankingsequenceofpri-miR-9waspredictedwith
TFSEARCH(http://www.cbrc.jp/research/db/TFSEARCH.html).
Statisticalanalysis
Thestatisticalsignificanceofdifferencesbetweengroupswasdetermined
bytheStudentttestandtwo-wayANOVA.Forsurvivalanalysis,the
Kaplan–Meiermethodandlog-ranktestwereperformed.Correlations
betweenvariablesweredeterminedbyaSpearmancorrelationcoefficient.
AllanalyseswereperformedusingSPSS16.0software.Differenceswere
consideredsignificantatapvalueof,0.05.
Studyapproval
AllanimalprotocolswereapprovedbytheInstitutionalCommitteeonthe
UseofAnimalsforResearchandTeaching.Allhumansampleswereob-
tainedwithinformedconsentinaccordancewiththeregulationsapproved
bytheAffiliatedPeople’sHospitalofJiangsuUniversity.
Results
Runx1isinvolvedinthedifferentiationandfunctionofMDSCs
Runx1isanessentialtranscriptionfactorthatcontrolsthedevel-
opmentofmultiplehematopoieticlineageexpressionandmodu-
latesseveralgenesunderlyingmyeloiddifferentiation(24).In
ourexperiments,G-MDSCsandM-MDSCswereisolatedfrom
spleensofmicebearingLLCforflowcytometricanalysis,andthe
ratioofG-MDSCstoM-MDSCswas～3:1(SupplementalFig.2A,
2B).Wepreviouslyshowedthatb-glucanscouldpromotethe
differentiationofM-MDSCsintomorematuratemyeloidcells
withreducedimmunosuppressivefunctions.Inthepresentstudy,
wefurtherfoundthatWGPscouldalsodownregulatethesup-
FIGURE4.DownregulationofmiR-9inMDSCspromotesthedifferentiationofthecellsandimpairstheirsuppressivecapacity.IsolatedM-MDSCsand
G-MDSCsfromtumor-bearingmicespleensweretransfectedwith100nMmiR-9mimics(miR-9),200nMmiR-9inhibitors(miR-9i),ormiRNAcontrol;
after24h,(A)relativeexpressionofmiR-9wasdetectedbyqRT-PCR.(B)M-MDSCstransfectedwithmiR-9inhibitorsornegativecontrolwerestained
withspecificAbsagainstCD11c,F4/80,CD40,CD80,CD86,andMHCclassII(isotype:solidgray).(CandD)TransfectedM-MDSCs(C)andG-MDSCs
(D)wereharvested,andarginaseactivityandROSlevelsweremeasuredasdescribedinMaterialsandMethods.RNAfromeachgroupwasextractedand
reversetranscribedforqRT-PCRforiNOS.(EandF)M-MDSCs(E)andG-MDSCs(F)weretransfectedwithmiR-9mimics,miR-9inhibitors,ormiRNA
control;after24h,cellswereharvestedandthencoculturedwithrespondercells,CD4
+
Tcells(left),orCD8
+
Tcells(right)(MDSC/Tcellratioof2:1,1:1,
0.5:1)inthepresenceofanti-CD3mAbandanti-CD28mAbfor72h.SuppressivefunctionofMDSCsonTcellproliferationwasmeasuredbyincor-
porationof[
3
H]thymidine.StatisticalsignificanceisevaluatedbetweenmiR-9/miR-9igroupandcontrolgroup.Dataareshownasmean6SDofthree
independentexperiments.p,0.05,p,0.01,p,0.001.Ctrl,control.
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pressiveeffectofG-MDSCs(SupplementalFig.2C–F).Notably,
Runx1expressioninM-MDSCsandG-MDSCswasupregulated
afterb-glucanstimulation(Fig.1A).KnockdownofRunx1in
MDSCsbysiRNAsignificantlyblockedtheb-glucan–mediated
effect.AsshowninFig.1C,WGP-inducedM-MDSCdifferenti-
ationwaspartiallyblockedupontheinhibitionofRunx1expres-
sion.LevelsofCD11c,F4/80,CD40,CD80,CD86,andMHC
classIIexpressionweredecreasedupondownregulationofRunx1
expression.Furthermore,levelsofarginase,iNOS,andROSse-
cretedbyM-MDSCsandG-MDSCsweremarkedlyupregulated
afterRunx1wasknockeddown(Fig.1D,1E).Additionally,
knockingdownRunx1expressioninbothM-MDSCsand
G-MDSCssignificantlyenhancedthecapacityofMDSCsto
suppressCD4
+
andCD8
+
Tcellproliferation(Fig.1F,1G).Thus,
thesedatasuggestthatRunx1isinvolvedinregulatingthedif-
ferentiationandfunctionofMDSCs.
ExpressionofmiR-9isdecreasedinMDSCsafterWGP
treatment
TocharacterizethedifferentialexpressionofmiRNAsinMDSCs
uponWGPstimulation,wefirstperformedmiRNAmicroarray
analysisonRNAsamplesisolatedfromMDSCstreatedwithor
withoutWGPs.Itwasfoundthat61miRNAsweredownregulated
whereas40miRNAswereupregulatedinMDSCsstimulatedby
WGPs.ToidentifythepossiblemiRNAsinvolvedinthediffer-
entiationandfunctionofMDSCs,weusedTargetScan6.1and
miRandasoftwaretopredictmiRNAsthatcouldbindtothe39UTR
ofRunx1.AccordingtothedatafrommiRNAmicroarraysand
predictivesoftwareanalysis,threemiRNAsthatweredownregu-
latedinMDSCsuponWGPstimulationandcouldalsobindto
Runx139UTRwereselected:miR-9,miR-181d,andmiR-181b.
WeconfirmedtheexpressionofthesemiRNAsbyqRT-PCR
analysis,amongwhichtheexpressionofmiR-9andmiR-181d
wasconsistentwiththedatafrommicroarray(Fig.2A).Tofurther
verifytheexpressionofmiR-9andmiR-181dinM-MDSCsand
G-MDSCsafterWGPstimulation,wefoundthatmiR-181dex-
pressionwasdecreasedinG-MDSCsbutupregulatedinM-MDSCs
(Fig.2B).However,onlymiR-9wasdecreasedinbothM-MDSCs
andG-MDSCsstimulatedbyWGPs(Fig.2C).Toverifywhetherthe
reductionofmiR-9wasmediatedviathedectin-1pathway,wefound
thatmiR-9expressionwasreversedtohighlevelsafterblocking
dectin-1withanti–dectin-1Ab(Fig.2D).Takentogether,thesedata
suggestthatmiR-9maybeinvolvedintheregulationofMDSCdif-
ferentiationandfunction.
miR-9directlytargetsRunx1inMDSCs
Next,weinvestigatedwhetherRunx1wasadirecttargetofmiR-9.
UsingTargetScan6.1combinedwithmiRandaanalyses,wefound
thatthe39UTRoftheRunx1genehadtwohighlyconservedseed
sequences(position185–190andposition653–658inmice)for
miR-9(Fig.3A).Becausethereexistedtwoseedsequencesinthe
Runx139UTR,wefirsttestedthecontributionofeachseedse-
quenceforregulatingtheexpressionofRunx1.MouseRunx1
39UTR(1+2)containingposition185–190(Runx139UTR1)and
FIGURE5.miR-9reversestheregulationofdiffer-
entiationandfunctiononMDSCsbyWGPs.Isolated
M-MDSCsandG-MDSCsfromtumor-bearingmice
spleensweretreatedwithWGPsat100mg/ml,and
thencellsweretransfectedwithmiR-9mimics(miR-9)
ormiRNAcontrol.(A)M-MDSCswerestainedwith
specificAbsagainstCD11c,F4/80,CD40,CD80,
CD86,andMHCclassII(isotype:solidgray)after
48h.(BandC)M-MDSCs(B)andG-MDSCs(C)were
harvestedtoanalyzethearginaseactivity,iNOSmRNA
expression,andROSlevel.Dataareshownasmean6
SDofthreeindependentexperiments.p,0.05,
p,0.01.Ctrl,control.
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position653–658(Runx139UTR2)ormutatedversions(Runx1
39UTR1mutantwithWTseedsequence653–658[Runx139UTR1
Mut+2]andRunx139UTR2mutantwithWTseedsequence
185–190[Runx139UTR2Mut+1])(Fig.3B)wereclonedinto
apsiCHECKluciferasereportervectordownstreamfromRenilla
luciferaseandthenusedtoassesswhethermiR-9couldrepress
luciferasegeneexpressionin293Tcells.AsshowninFig.3C,
luciferaseexpressionincellstransfectedwithRunx139UTR
(1+2)wasrepressedbymiR-9.Moreover,mutationofeitherpo-
sition185–190orposition653–658intheRunx139UTRdidnot
significantlyaffecttheluciferaseexpressioninthepresenceof
miR-9,furtherindicatingthatbothseedsequencesregulatedthe
expressionofRunx1.Themutationofbothposition185–190and
position653–658ofthe39UTRseedmatchsequencesrelievedthe
inhibitionbymiR-9.Thesedatasuggestthatbothseedsequences
ofRunx139UTRplayanimportantroleinregulatingtheex-
pressionofRunx1.TodeterminewhetherbindingofmiR-9to
Runx139UTRcouldregulateRunx1expression,amonocyte/
macrophagecelllineRAW264.7wastransfectedwithmiR-9
mimicsatdifferentdoses.TransfectionwasconfirmedusingqRT-
PCR(Fig.3D),andRunx1expressionwasmeasuredatboth
mRNAandproteinlevels.AsshowninFig.3Eand3F,theprotein
levelsofRunx1inRAW264.7cellstransfectedwithmiR-9
mimicswerelowerthaninnegativecontroltransfectedcells,
whereasthetranscriptionlevelofRunx1wasnotaffected.Nota-
bly,Runx1siRNAnotonlydownregulatedthetranscriptionbut
alsoproteinlevelsofRunx1.Takentogether,theseresultsdem-
onstratethatmiR-9directlytargetsRunx1todownregulateRunx1
expression.
DownregulationofmiR-9promotesMDSCsdifferentiationand
impairstheirsuppressivecapacity
TodefinethefunctionofmiR-9inMDSCdifferentiationand
function,M-MDSCsandG-MDSCsweretransfectedwithmiR-9
mimicsorinhibitors.AsshowninFig.4,downregulationofmiR-9
inM-MDSCscouldenhancetheexpressionofCD11c,F4/80,
CD40,CD80,CD86,andMHCclassII,indicatingamature
phenotypeofMDSCsuponmiR-9inhibition.Furthermore,the
suppressivefactorssecretedbyM-MDSCsandG-MDSCs,includ-
ingarginase,iNOS,andROS,weredecreasedaftermiR-9was
knockeddown.Incontrast,overexpressionofmiR-9significantly
enhancedtheproductionofthesesuppressivefactorsbyMDSCs
(Fig.4C,4D).Todeterminewhetherthesuppressivecapacityof
MDSCswasregulatedbymiR-9,isolatedsplenicM-MDSCsand
G-MDSCsfrommicebearingLewislungcarcinomaweretrans-
fectedwithmiR-9mimicsorinhibitors.AsshowninFig.4Eand
4F,miR-9mimicsremarkablyenhancedtheabilityofMDSCsto
suppressCD4
+
andCD8
+
TcellswhereasinhibitionofmiR-9re-
ducedtheirsuppressivefunction.Thus,theseresultsdemonstrate
thatdownregulationofmiR-9promotesMDSCsdifferentiationand
impairstheirsuppressivecapacity.
ExogenousmiR-9reversestheregulatoryeffectofWGPson
MDSCdifferentiationandfunction
TofurtherconfirmthatmiR-9regulatesthedifferentiationand
functionofMDSCs,exogenousmiR-9wassupplementedtocul-
turedMDSCsinthepresenceofWGPs.ItwasfoundthattheWGP-
inducedeffectonMDSCswasreversedbyupregulationofmiR-9.
AsshowninFig.5A,levelsofCD11c,F4/80,CD40,CD80,
CD86,andMHCclassIImoleculeonMDSCswerereducedafter
miR-9wasenhanced.Moreover,botharginaseactivityandlevels
ofiNOSandROSwerealsoenhancedtogetherwiththeincreased
miR-9inMDSCs(Fig.5B,5C).Therefore,thesedatasuggestthat
miR-9inhibitstheregulatoryeffectofWGPsonMDSCdiffer-
entiationandfunction.
DownregulationofmiR-9inMDSCssuppressestumorgrowth
invivo
ToexaminethefunctionofmiR-9inMDSCsinvivo,weestablished
amousemodelwithLLCandintratumorouslyinjectedmiR-9
mimic–orinhibitor–modifiedMDSCstoobservetumorprogres-
sion.AsshowninFig.6A,tumorsinmicetreatedwithmiR-9
FIGURE6.DownregulationofmiR-9inMDSCsdelaystumordevelopment.MurineLLCcells(3310
6
)wereinjectedintoC57BL/6mice.(AandB)Then,
sortedMDSCsfromtumor-bearingmicespleensweretransfectedwith100nMmiR-9mimics(miR-9agomir-MDSC),200nMinhibitors(miR-9antagomir-
MDSC),ornegativecontrol(Ctrl-MDSC),andthesemodifiedMDSCs(2310
6
permouse)wereinjectedintothetumorsofmiceafterpalpabletumorswere
formed.Fivedayslater,themodifiedMDSCswereadministeredforthesecondtime.(A)Tumorsweremeasuredwithacaliperatindicatedtime(n=8)and(B)
miceineachgroupweremonitoredforsurvivaluntil48daftertumorchallenge(n=10).(C–E)OnemicrogrammiR-9mimics(miR-9agomir),2mginhibitors
(miR-9antagomir),ornegativecontrolwasinjectedintotumorsofmiceafterpalpabletumorswereformed.Fivedayslater,eachgroupwasadministeredfor
thesecondtime.(C)Tumorsweremeasuredwithacaliperatindicatedtime(n=8)and(D)miceineachgroupweremonitoredforsurvival(n=10).(E)
Arginaseactivityofcellsfromtumortissueswasmeasured.Resultsareexpressedasmean6SD.p,0.05,p,0.01.Ctrl,control.
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inhibitor(antagomir)–modifiedMDSCsgrewslowerthandid
micetreatedwithcontroltransfectedMDSCs.Conversely,tumors
inmicetreatedwithmiR-9mimic(agomir)–transfectedMDSCs
werefoundtogrowfasterthanthoseinmicetreatedwithcontrol
transfectedMDSCs.Moreover,tumor-bearingmicetreatedwith
miR-9mimic–modifiedMDSCsshowedsignificantlyreducedsur-
vivalwhencomparedwiththecontrolgroup.However,therewasno
obviousprolongedsurvivalinmiR-9inhibitor–modifiedMDSCs
(Fig.6B).TofurtherdeterminewhethermiR-9exertsanyeffecton
tumorgrowth,miR-9mimicsorinhibitorsweredirectlyinjected
intotumors.WefirstconfirmedthatmiR-9mimicsorinhibitors
couldcrossthemembraneofMDSCs(SupplementalFig.1I).As
showninFig.6Cand6D,upregulationofmiR-9significantlypro-
motedtumorgrowth.However,downregulationofmiR-9didnot
showanysignificanteffectsonsurvivalrate.Interestingly,miR-9
mimicssignificantlyincreasedthelevelofarginaseintumortissues
whereasmiR-9inhibitorsreducedarginasemRNAexpression(Fig.
6E).SimilareffectswerealsoobservedintheCT26coloncarcinoma
model(SupplementalFig.3).
CREBcontrolstheexpressionofmiR-9
CREBisatranscriptionfactorthatregulatesvariouscellular
responses,includingproliferation,differentiation,andsurvival
(25–27).CREBisinducedbymanygrowthfactorsandinflam-
matorysignalsandsubsequentlymediatesthetranscriptionof
genescontainingacAMP-responsiveelement(28).Inourstudy,
wefoundthatCREBexpressionwassignificantlydecreasedin
bothM-MDSCsandG-MDSCsuponWGPstimulation(Fig.7A).
Usinganti–dectin-1Abtoblockthedectin-1pathwayfurther
confirmedthatWGP-inducedCREBdecreasewasmediatedby
thedectin-1pathway.AsshowninFig.7B,CREBexpressionwas
recoveredtoahighlevelafterinhibitionofthedectin-1pathway.
Interestingly,afterCREBinMDSCswasknockeddown(Fig.7C),
miR-9levelswerealsosignificantlydecreased(Fig.7D).Tofur-
therdelineatetherelationshipbetweenCREBandmiR-9,we
foundthepotentialbindingsiteforCREBwithinthemiR-9
promoterregionusingTFSEARCHforprediction.Thus,we
establishedtheWTandMutofluciferasereportervectorscon-
tainingthepri-miR-9promoterregion.Thepredictivebindingsite
forCREBwas59-TGACGTCA-39,andthemutatedbindingsite
waschangedto59-AATTCCGG-39(Fig.7E).Wenextcotrans-
fectedCREBoverexpressionplasmidsorcontrolplasmid
pcDNA3.1,WT-miR-9promoterfireflyluciferasereportervectors,
ormutantmiR-9promotervectortogetherwiththymidinekinase
promoterRenillaluciferasereporter(pTK-RL)plasmidsinto
293Tcells.AsshowninFig.7F,ectopicexpressionofCREB
FIGURE7.miR-9isunderthecontrolof
CREB.(A)SortedM-MDSCsandG-MDSCs
wereculturedinthepresenceorabsenceof
WGPsat100mg/mlfor24h.qRT-PCRwasused
todetectCREBmRNAexpression.(B)Thepu-
rifiedM-MDSCsandG-MDSCswerepretreated
withanti–dectin-1mAborratIgG(5mg/ml)for
1hat37?Candthentreatedwith100mg/ml
WGPs.After24hstimulation,cellsweresub-
jectedtoanalyzeCREBmRNAlevels.(CandD)
IsolatedM-MDSCsandG-MDSCsweretrans-
fectedwithCREBsiRNA(siCREB)ornegative
controlfor24h,andrelativeexpressionof
CREB(C)andmiR-9(D)wasanalyzedbyqRT-
PCR.(E)Constructionofluciferasereporter
vectorscontainingpri-miR-9promoterregion.
ThebindingsiteforCREB(59-TGACGTCA-39)
waspredictedaccordingtoTFSEARCHbrowser,
andthemutationtypewasmutatedbyPCR-based
mutagenesis(59-AATTCCGG-39).(F)293Tcells
(1310
5
)werecotransfectedwithWTormutant
pGL3–miR-9promoterfireflyluciferasereporter
vectors,thymidinekinasepromoterRenillalucif-
erasereporter(pTK-RL)plasmids,togetherwith
CREBoverexpressionplasmidsorcontrolplas-
mids(pcDNA3.1).After24h,fireflyluciferase
activitywasmeasuredandnormalizedbyRenilla
luciferaseactivity.Dataareshownasmean6SD
ofthreeindependentexperiments.p,0.05,
p,0.01.Ctrl,control.
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enhancedthetranscriptionalactivityofthe59flankingsequenceof
miR-9-1,andmutationofthebindingsiteforCREBabolishedthe
enhanciveeffectofCREB.Thus,thesedatasuggestthatmiR-9
expressionisdirectlyregulatedbyCREB.
HighlevelsofmiR-9inverselycorrelatewithRunx1expression
inlungcancerpatients
WefurtherevaluatedmiR-9expressionanditscorrelationwith
Runx1inhumanlungcancers.First,wefoundmarkedlyincreased
numbersofMDSCsintumortissues(lungcancer)whencompared
thoseindistalnontumoroustissues(distallung)(Fig.8A).Fur-
thermore,miR-9expressionintumortissueswassignificantly
higherthancontrols.However,therewasnoobviouselevationof
miR-9inplasmaoflungcancerpatientswhencomparedwith
healthyindividuals(Fig.8B).AsshowninFig.8C,expression
ofRunx1wasextremelylowintumortissues,andtherewas
amodestcorrelationbetweenRunx1andmiR-9intumortissues
(Fig.8E).Additionally,tumortissuesexpressedhighlevelsof
arginase(Fig.8D),andarginasehadnegativecorrelationwith
Runx1butpositivecorrelationwithmiR-9(Fig.8E).Allofthese
dataindicatethatmiR-9ishighlyexpressedinlungcancersandis
associatedwithMDSC-derivedsuppressivefactors.
Discussion
Inthisstudy,weshowthatmiR-9regulatesMDSCdifferentiation
andfunctionbytargetingRunx1expression.Downregulationof
miR-9couldsignificantlypromotethedifferentiationofMDSCs
andimpairtheirsuppressivecapacity.Moreover,miR-9levelin
MDSCswascontrolledbyCREBtranscriptionfactor.Takento-
gether,ourresultshavedemonstratedakeyroleofmiR-9in
regulatingthedifferentiationandfunctionofMDSCs.
RecentstudieshaveshownthatmiRNAscontrolmyeloidcell
proliferation,differentiation,maturation,andactivation(29).Ithas
beenreportedthatmiRNAs,includingmiR-17-5p,miR-20a(30),
miR-223(31),andmiR-494(32),areinvolvedinregulatingthe
differentiationandfunctionoftumor-expandedMDSCs.miR-17-
5pandmiR-20aalleviatetheimmunosuppressivepotential
ofMDSCsbymodulatingSTAT3expression(30).Additionally,
miR-223suppressesdifferentiationoftumor-inducedMDSCs
frombonemarrowcellsviatargetingMEF2C.Moreover,miR-494
promotestheaccumulationandfunctionsoftumor-expanded
MDSCsviaregulatingPTENexpression.However,themolecular
eventscontrollingthedifferentiationandmaturationoftumor-
expandedMDSCsarelargelyunknown.
miR-9hasbeencharacterizedasanessentialelementinregu-
latingimmuneresponses(33),neuronaldifferentiation(34),
posttraumaticstress(35),andvariouscancers(36,37).Our
previousstudieshavedemonstratedthattheimmunomodulator
b-glucancouldpromoteMDSCdifferentiationintomoremature
myeloidcellsandinhibittheregulatoryfunctionofMDSCs.Inthe
presentstudy,wefoundthatmiR-9expressionwassignificantly
downregulatedinMDSCsstimulatedbyWGPs.Moreover,inhi-
bitionofmiR-9inM-MDSCsupregulatedtheexpressionof
CD11c,F4/80,CD40,CD80,CD86,andMHCclassIImolecule,
indicatingthatdownregulationofmiR-9levelinM-MDSCscould
promotedifferentiationofM-MDSCsintoamorematurestate.
Additionally,afterinhibitionofmiR-9inbothM-MDSCsand
G-MDSCs,levelsofsuppressivefactors,includingarginase,
iNOS,andROS,weresignificantlydecreased.Notably,thecapacity
ofMDSCstosuppressCD4
+
orCD8
+
Tcellproliferationwasre-
ducedwhenthesecellsweretransfectedwithmiR-9inhibitors.On
thecontrary,upregulationofmiR-9inMDSCssignificantlyenhanced
FIGURE8.miR-9isincreasedinhumanlungcancer.(A)MDSCs(arrow,yellowspots)accumulationintumortissuesordistalnoncanceroustissuesof
lungcancerswereanalyzedbyimmunofluorescentstainingwithFITC-labeledanti-CD11bmAbandallophycocyanin-labeledanti-CD33mAb(original
magnification3200).ThenumberofMDSCswasquantified.(B)miR-9relativeexpressionwasdetectedintumortissuesanddistalnoncanceroustissues
(n=18).Plasmafrom15healthypersonsandlungcancerpatientswascollectedtodetectmiR-9levels.(CandD)Runx1(C)andarginase(D)mRNA
expressionwasanalyzedintumortissuesordistalnoncanceroustissues.(E)CorrelationofmiR-9,Runx1,andarginasebetweeneachotherwasanalyzed.
Resultsareexpressedasmean6SD.p,0.05,p,0.01,p,0.001.
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thesuppressiveeffectofthecells.Thus,theseresultshavedemon-
stratedthatmiR-9playsacriticalroleinmodulatingMDSCdiffer-
entiationandfunction.TofurtherdefinearoleofmiR-9inthe
differentiationandfunctionofMDSCs,wefoundthattheWGP-
inducedeffectonMDSCswasreversedwhenmiR-9wasexoge-
nouslysupplementedinculture.Consistently,ourinvivoexperi-
mentsfurthershowedthatmiR-9inhibitor–modifiedMDSCs
exhibitedpoorsuppressivecapacity,resultinginsuppressedtumor
growthwhereasMDSCswithhighmiR-9expressionpromoted
tumordevelopmentinvivo.Thus,thesedatademonstratethat
miR-9isacriticalregulatorinMDSCdifferentiationandfunction.
Inthisstudy,weidentifiedRunx1asafunctionaltargetofmiR-9
intheregulationofMDSCdifferentiationandfunction.Runx1is
awell-knowntranscriptionfactorduringtheformationofhemo-
poieticstemcellsfromthehemogenicendothelium(38–40),which
isconsideredasanimportantdifferentiationinducer.Runx1is
requiredforhemopoieticstemcelldifferentiationintoendothelial
cellsandpromotesthedifferentiationofmyeloid,lymphoid,and
megakaryocyticlineages(41)bycontrollingtheexpressionof
severalgenesregulatingmyeloiddifferentiation,includingGM-
CSF(42),myeloperoxidase(43),IL-3(44),aswellasM-CSFR
(45).Inthisstudy,wefoundthatRunx1wasmarkedlyupregulated
inMDSCsstimulatedbyWGPs.However,afterknockingdownthe
expressionofRunx1bysiRNA,theWGP-inducedeffectwaspar-
tiallyblocked.Theexpressionlevelsofcostimulatorymolecules
(CD40,CD80,CD86)andMHCclassIImoleculeonM-MDSCs
werereducedafterknockingdownRunx1,whichindicatesthat
Runx1isinvolvedinthedifferentiationofM-MDSCs.Additionally,
suppressivefactorssecretedbyMDSCs,includingarginase,iNOS,
andROS,wererestoredtohighlevelsinWGP-treatedMDSCswith
knockingdownRunx1,andthecapacityofMDSCstosuppress
CD4
+
andCD8
+
TcellswasenhancedafterknockingdownRunx1,
whichsuggeststhatRunx1alsoplaysanessentialroleinregulat-
ingthefunctionofMDSCs.Furthermore,ourresultsrevealedthat
Runx139UTRhadtwo“seedregions”formiR-9andthatmiR-9
canbindtothesetworegionstoregulatethetargetgeneexpression.
AnotherinterestingfindinginourstudyisthatmiR-9expression
iscontrolledbyanothertranscriptionfactor,thatis,CREB.CREB
isamemberoftheactivatingtranscriptionfactor/CREBfamilyof
transcriptionfactors,whichcanregulatediversecellularresponses,
includingproliferation,survival,anddifferentiation(25–27).Forin-
stance,CREBhasbeenreportedtobeaproto-oncogene,leadingto
increasedproliferationofmyeloidcellsandmyeloproliferativedis-
eases(46,47).Inthepresentstudy,weexcitinglyfoundthatCREB
levelwasdownregulatedinMDSCsafterWGPtreatment,whichwas
dectin-1–dependent.Moreinterestingly,afterknockingdownCREB
bysiRNA,thelevelofmiR-9wasalsodecreased.Thedatamadeus
questionwhethermiR-9expressionwasregulatedbyCREB.Inter-
estingly,thepredictivesoftwareshowedthatCREB’sbindingsite
wasinmiR-9promoterregion.AsindicatedinFig.7F,theluciferase
reporterassayfurtherconfirmedthepredictionanddemonstratedthat
CREBcouldbindtothepromoterofmiR-9andregulateitsex-
pression.
Effectivecancerimmunotherapyrequirestheelicitationofpo-
tentantitumorTcellresponsesandeliminationofimmunosup-
pressivecellpopulationssuchasMDSCsandregulatoryTcells.As
thesuppressivefunctionofMDSCsiscloselyassociatedwiththeir
immaturestate,theinductionofMDSCdifferentiationcouldbean
effectiveapproachinreversingtheirsuppressivecapacitytonormal
immuneresponses.Thus,promotingthedifferentiationofMDSCs
andinhibitingtheirsuppressivefunctionappeartobeeffective
strategiestotargetMDSCsincancerimmunotherapy.Inthisstudy,
wefirstdemonstratedthatmiR-9couldregulatethedifferentiation
andfunctionofMDSCsbytargetingRunx1.Downregulationof
miR-9couldpromotethedifferentiationofMDSCsandimpair
theirsuppressivecapacity.Importantly,downregulatingmiR-9
levelinMDSCscouldsignificantlydelaythetumordevelop-
mentinthemouseLLCmodel.Moreover,theclinicaldataalso
showedthathighlevelsofmiR-9wereobservedintumortissues.
Furthermore,miR-9positivelycorrelatedwitharginasewhereas
Runx1negativelycorrelatedwitharginase.Althoughtumor-bearing
micetreatedwithmiR-9inhibitors(antagomir)–modifiedMDSCs
showedreducedtumorgrowth,thedirectinjectionofmiR-9
inhibitorsintotumorsdidnotshowanysignificanteffectonsup-
pressingtumorgrowth.Furtherstudiesarewarrantedtoestablishan
optimizedprotocolforprolongingthesurvivaloftumor-bearing
micetreatedwithmiR-9antagomir–modifiedMDSCsandtode-
terminewhetherthedirectinjectionofmiR-9inhibitorsmayrep-
resentaneffectivestrategyforantitumortherapy.
Insummary,ourresultsindicatethattargetingofmiR-9can
reduceMDSC-mediatedsuppressionandthenenhanceantitumor
immunity,whichmightbefurthervalidatedasapotentialthera-
peutictarget.
Acknowledgments
WethankDr.MiaomiaoZhang,ShengXia,MinYang,andHaiyanYoufor
helpfuldiscussionsandtechnicalassistance.
Disclosures
Theauthorshavenofinancialconflictsofinterest.
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