RolesofdifferentialexpressionofmicroRNA-21-3pandmicroRNA-433inFSHregulationinratanteriorpituitarycellsDong-XuHan1,Xu-LeiSun1,Ming-QiangXu1,...
Dong-XuHan1,Xu-LeiSun1,Ming-QiangXu1,Cheng-ZhenChen1,HaoJiang1,
YanGao1,BaoYuan1,Jia-BaoZhang1
1DepartmentofLaboratoryAnimals,CollegeofAnimalSciences,JilinUniversity,Changchun,Jilin,P.R.China
Correspondenceto:Jia-BaoZhang,email:zjb515@126.com
BaoYuan,email:yuanbao1982@163.com
Keywords:ratanteriorpituitarycell,FSHsecretion,miR-21-3p,miR-433,animalreproduction
Received:February06,2017Accepted:March11,2017Published:March28,2017
ABSTRACT
INTRODUCTION
Thepituitary,whichisthemostimportantendocrineorganinanimals,functionsasamainregulatorofnumerousphysiologicalprocessesthroughitscontrolofdownstreamendocrineglands[1].Seventypesofhormonessecretedfromthepituitaryplayimportantrolesintheregulationoforganismalactivities[2].Folliclestimulatinghormone(FSH),oneofthegonadotropinhormones(GTH),isapivotalregulatorofreproduction[3].FSHregulatestheproductionofseveralgrowthfactorsthatplayavitalroleinearlyfolliculogenesisandanimareproduction[4–6].
FSH,aglycoproteinhormoneencodedbytheFSHbgene[7],comprisestwosubunits,specificallyacommonαsubunitandauniqueβsubunit,whichareinvolvedinspecificbiologicalactivities[3,7].Inmales,FSHregulatesspermatogenesis,andinfemales,thishormoneisindispensableforoogenesis[3].FSHbindstothefolliclestimulatinghormonereceptor(FSHR)locatedonSertolicellsofthetestisandgranulosacellsoftheovariestotransmititssignalandexertitsfunctions[8].NumerousstudieshaveindicatedthatFSHsecretionisregulatedbymanyfactors.Lowerexpressionlevelsofgonadotropinreleasinghormonereceptor(GnRHR)canstimulatemaximalproductionofFSH[9],andatthetranscriptionallevel,FSHsecretionismainlyregulatedbythegonadotropinreleasinghormone(GnRH)signalingpathway[10].Furthermore,thegeneticlinkageofnovelsingle-nucleotidepolymorphisms(SNPs)withinbovineFSHbcaninfluencetheserumFSHconcentrations[11].
ThemiRNAsareafamilyofshort,non-codingRNAsthatcanplaycrucialrolesinbothanimalsandplants[12,13].miRNAscancombinewiththemRNAsofprotein-codinggenestodecreasethetranslationalefficiencyormodulatethepost-transcriptionallevelsofthemRNAs[14].NumerousmiRNAshavebeenidentifiedinnearlyallmetazoangenomesexaminedsincethediscoveryofthetwofirstmiRNAs,lin-4andlet-7[12,15,16],andmanystudieshavereportedthatmiRNAscaninfluencehormoneregulation.Forexample,miR-26bupregulatesthegrowthhormonelevelsbytargetinglymphoidenhancerbindingfactor1(Lef-1)inGH3cells,whereasmiR-129-5p,miR-202andtwoothermiRNAsrepress】thehumangrowthhormonereceptor(GHR)expressionlevelsinbothnormalandcancercells[17,18].However,itremainsunclearwhetherFSHsecretionisregulatedbymiRNAs.
RESULTS
IdentificationofmiRNAsthatpotentiallytargettheFSHb3’UTR
ToidentifyratmiRNAsthatpotentiallytargetthe3’UTRoftheFSHbgene,weusedtheTargetScanprogramtopredict150miRNAsthatcouldtargetthe3’UTRoftheFSHbmRNAatthepost-transcriptionallevel(SupplementaryTable1).AfterdeletingtheduplicatetargetsitesandmiRNAsthatarenotexpressedintheratpituitarybasedontotheresultsofapreviousmicroarraystudy[2],weselected45preferredcandidatemiRNAs(Table1).TocomprehensivelyverifytheinteractionsbetweenthesemiRNAsandtheFSHbmRNA,weconstructedareporterplasmid,pmiR-FSHb-3’UTR-WT(SupplementaryFile2),inwhichtheFSHb3’UTRwascloneddownstreamofafireflyluciferasereportergene.Wethenusedascreeningsystembasedontheluciferasereporterplasmidcarryingthefull-length3’UTRoftheFSHbmRNAandfoundthat18miRNAs,specificallymiR-433-3p,miR-323-3p,miR-328a-3p,miR-3573-3p,miR-204-5p,miR-206-5p,miR-31a-5p,miR-7a-5p,miR-880-3p,miR-186-5p,miR-503-5p,miR-383-5p,miR-324-5p,miR-505-5p,miR-27b-3p,miR-221-5p,miR-320-3pandmiR-21-3p,couldsuppresstheexpressionofthereporterbymorethan30%(Figure1).
DetectionoftheexpressionoftheidentifiedmiRNAsatdifferentdevelopmentalstages
ValidationoftheinteractionbetweenthemiRNAsandFSHbinvitro
ToexaminewhetherthesilencingofFSHbismediatedbythespecific,directinteractionofmiR-21-3pandmiR-433withtheFSHbtargetsite,wepredictedtherespectivetargetpositionsofthetwomiRNAsintheFSHb3’UTRusingtheTargetScanprogram(Figures3Aand3B),andwethenmutatedthecomplementarysitesofthetwomiRNAseedregionstogeneratepmiR-FSHb-3’UTRMUTandpmiR-FSHb-3’UTR-MUT1(SupplementaryFile3).WethencotransfectedmiR-21-3pmimicsandpmiR-FSHb-3’UTR-WTinto293Tcells,whichledtoa~60%reductioninluciferaseactivity;interestingly,cotransfectionofthemiR-21-3pmimicsandpmiRFSHb-3’UTR-MUTinto293Tcellsyieldednosignificantchangesinluciferaseactivity(Figure3C).Similarly,cotransfectionofmiR-433andpmiR-FSHb-3’UTR-WTinto293Tcellsyieldedamorethana30%decreaseinluciferaseactivity,whereascotransfectionofmiR-433andpmiR-FSHb-3’UTR-MUT1yieldednochangesinluciferaseactivity(Figure3D).Therefore,theresultssuggestedthatmiR-21-3pandmiR-433canregulatetheexpressionofFSHb.
EffectofmiR-21-3pandmiR-433transfectiononratprimarypituitarycells
TodetectthegrowthofculturedprimarypituitarycellsaftertransfectionwithmiR-21-3pandmiR-433,weobservedthegrowthofthepituitarycellsandnotedthattheyappearedtobeingoodcondition(Figures4A-4D).Todeterminethetransfectionefficiencyachievedwitha100nMconcentrationofthenegativecontrols,mimics,inhibitornegativecontrolsandinhibitorsofmiR-21-3p
andmiR-433,wemeasuredtheexpressionlevelsofthetwomiRNAs,andtheresultsshowedthattransfectionofthemiR-21-3pmimicsignificantlyincreasedtheexpressionlevelofmiR-21-3p,whereastransfectionofthemiR-21-3pinhibitorsignificantlydecreasedtheexpressionlevelofmiR-21-3p(Figure4E).WeobservedasimilarpatternformiR-433:theexpressionlevelwassignificantlyincreasedinthemimicgroupandsignificantlydecreasedintheinhibitorgroup(Figure4F).Theseresultsindicatedthatthetransfectionwassuccessful.
miR-21-3p-andmiR-433-mediatedregulationoftheFSHbexpressionlevelsinandFSHsecretionbyratprimarypituitarycells
ToverifythatbothmiR-21-3pandmiR-433targettheFSHbgeneandtogainfurtherinsightsintotheirregulationofreproduction,wemeasuredtheexpressionlevelsofFSHbinratprimaryanteriorpituitarycellsbyquantitativeRT-PCRandthesecretionofFSHbythesecellsthroughELISA.
TheseresultsdemonstratedthatbothmiR-21-3pandmiR-433canregulateFSHbexpressionbydirectlybindingtotheFSHb3’UTRandinhibitingtheFSHbexpressionlevelsthroughthedegradationofmRNAandinhibitionoftranslation.
DISCUSSION
miRNAsarepost-transcriptionalregulatorsthatplaysignificantrolesincancer,disease,growthanddevelopment,andstemcelldifferentiation[19–21].Inaddition,miRNAsareexpressedinatissue-specific,time-dependentmannerandcontrolthedifferentiationormaintenanceoftissueidentity[22].Furthermore,miRNAsexerteffectsonthepituitary,includingbothpituitaryadenomasandthenormalpituitary.Inpituitaryadenomas,themiRNAprofilesmightprovidepotentialmarkersforpredictingthepituitaryadenomahistotypes[23].PreviousstudieshaveshownthatmiRNAsareimportantemergingelementsinmanytypesofadenomas,suchasGH-secreting,PRL-secreting,andnon-functionalpituitaryadenomas[24–26].Inaddition,miRNAscanaffectthedevelopmentofthenormalpituitary.Forexample,in2010,ZichaoZhangetal.reportedthatmiR-26bregulatestwomainfactors,Lef-1andpituitary-specificpositivetranscriptionfactor
pairedboxproteinPax-6(PAX6)[48].Nevertheless,theroleofmiR-433inpituitaryadenomashasnotbeeninvestigated.Regardinghormonesecretion,in2012,Riesteretal.reportedthatACTHstimulationcouldmodulatetheadrenalresponsebyinfluencingmiR-433toactasanendogenousmodulatoroftheglucocorticoidreceptor(Nr3c1)[49].SimilartomiR-21,theroleofmiR-433inpituitaryhormonesecretionremainslargelyunknownduetoalackofrelevantstudies.However,ourresultsshowthatmiR-433canregulatetheFSHbexpressionlevels,whichprovidesdataontheregulationofhormonesecretioninthepituitarybymiRNAs.
Takentogether,ourresultsshowthatbothmiR-21-3pandmiR-433down-regulateFSHbexpressionandcauseFSHsecretiondecreased.ThesefindingsprovideinsightsintotheeffectsoftheregulatorymechanismsofmiRNAsonthereproductivefunctionsofthepituitary.
MATERIALSANDMETHODS
Ethicsstatement
TheexperimentwasstrictlyconductedinaccordancewiththeguidelinesoftheGuidefortheCareandUseofLaboratoryAnimalsofJilinUniversity.TheprotocolwasapprovedbytheInstitutionalAnimalCareandUseCommitteeofJilinUniversity(PermitNumber:20160312).
Animalsandcellculture
TotalRNAwasextractedusingthemiRcutemRNAExtractionandSeparationKitandtheTRIzolreagent(Tiangen,Beijing,China)accordingtothemanufacturer’srecommendedprotocol.ThetotalquantityofRNAwasassessedwithaNanoDropND-2000spectrophotometer(NanoDropTechnologies).WethentransformedthetotalRNAintocDNAusingtheFastQuantRTKit(withgDNase)accordingtothemanufacturer’sinstructions(Tiangen,China).QuantitativeRT-PCRwassubsequentlyperformedwithaMastercyclerepRealplex2system(Eppendorf,Germany)andSuperRealPreMixPlus(SYBRGreen)accordingtothemanufacturer’sinstructions(Tiangen,China).ThemRNAandmiRNAprimersusedintheseassaysarelistedintheSupplementaryFile1.
TransfectionofmiRNAmimicsandinhibitors
AllmiRNAmimicsandinhibitorswerepurchasedfromGuangzhouRiboBioBiotechCo.,Ltd.Thetransfectionofratpituitarycellswasreferredtothepreviousstudy[55].Ratanteriorpituitarycellswereseededatadensityof3×105cellsperwellina24-wellplate.TransfectionwasperformedwithaLipofectamine3000TransfectionKit(ThermoFisherScientific,Waltham,USA)accordingtothemanufacturer’srecommendedprotocol.ThefinalconcentrationsofthemiRNAmimics,inhibitorsandnegativecontrolswere100nM.Aftertransfection,thecellswereincubatedfor24hforgeneexpressionanalysis.
ConstructionofthepmiR-FSHb-3’UTR-WTandpmiR-FSHb-3’UTR-MUTreporterplasmids
Thefull-length3’UTRoftheratFSHbmRNA(GenBankAccessionNo.NM_001007597.2)wasclonedbetweentheXhoIandNotIsitesofthepmiR-RBREPORTTMplasmid,generatingthepmiR-FSHb-3’UTRWTplasmid(SupplementaryFile2).Weintroducedsite-specificmutationsintothepmiR-FSHb-3’UTR-WTplasmidtointerruptthebindingsitesofmiR-21-3pandmiR-433,producingthepmiR-FSHb-3’UTR-MUTandpmiR-FSHb-3’UTR-MUT1plasmids(SupplementaryFile3).GuangzhouRibobioBiotechCo.,Ltd.,assistedintheconstructionofthereporterplasmids.Allconstructproductswereconfirmedviasequencing(RibobioBiotechCo.,Ltd.,Guangzhou,China).
Luciferasereporterassay
ToidentifythetargetmiRNAs,wecotransfected293Tcellswith45miRNAmimics,negativecontrols,pmiR-FSHb-3’UTR-WTandpmiR-RB-REPORTTM.FortheanalysisofmiR-21-3pandmiR-433,293Tcellswerecotransfectedwiththemimics,negativecontrols,pmiR-FSHb-3’UTR-WT,pmiR-FSHb-3’UTR-MUT,pmiR-FSHb-3’UTR-MUT1andpmiR-RB-REPORTTM.The293Tcellswereseededatadensityof1.5×104cellsperwellwith100μlofDMEMin96-wellplatesandtransfectedusingtheLipofectamine2000reagent(Invitrogen,USA).Aftertransfectionfor48h,theluciferaseactivitywasmeasuredusingafluorescenceintensitymeter(Veritas9100-002),andtheexperimentswererepeatedatleastthreetimes.Renillaluciferasewasusedasaninternalreferenceluciferasetominimizeexperimentalvariability.
FSHdetection
Wecollected50μlofthesupernatantfrompituitarycellsafterincubationfor24hfollowingtransfection.WemeasuredtheFSHlevelsunderthedifferentexperimentalconditionsusingaRatFSHELISAkitaccordingtothemanufacturer’sinstructions(HalingBiotechCo.,Ltd.,Shanghai,China).
Statisticalanalysis
Alldataareexpressedasthemeans±standarddeviationsfromthreeindependentexperiments.Significantdifferencesweredeterminedviaone-wayANOVAformultiplecomparisonsusingSPSS19.0forWindows.P<0.05wasconsideredstatisticallysignificant.
CONFLICTSOFINTEREST
Theauthorshavenoconflictsofinteresttodisclose.
FUNDING
ThisstudywassupportedbytheNationalNaturalScienceFoundationofChina(31501954)andtheKeyTechnologyR&DProgram(2015BAI07B02and20150101102JC).
Authorcontributions
B.Y.andJ.B.Z.wereresponsibleforthemainconceptionanddesignofthestudy;D.X.H.,X.L.S.,andM.Q.X.performedtheexperiments;D.X.H.,C.Z.C.,H.J.,andY.G.analyzedthedataandcontributedreagents;D.X.H.,B.Y.,andJ.B.Z.wrotethemanuscript;andalloftheauthorsapprovedthefinalversion.
REFERENCES
1.HongGK,PayneSCandJaneJA,Jr.Anatomy,Physiology,
andLaboratoryEvaluationofthePituitaryGland.
OtolaryngologicclinicsofNorthAmerica.2016;49:21-32.
2.YuanB,HanDX,DaiLS,GaoY,DingY,YuXF,Chen
J,JiangH,ChenCZandZhangJB.Acomprehensive
expressionprofileofmicrornasinrat'spituitary.
Internationaljournalofclinicalandexperimentalmedicine.
2015;8:13289-13295.
3.KumarTR,WangY,LuNandMatzukMM.Follicle
stimulatinghormoneisrequiredforovarianfollicle
maturationbutnotmalefertility.Naturegenetics.1997;
15:201-204.
4.MagalhaesDM,AraujoVR,Lima-VerdeIB,MatosMH,
SilvaRC,LucciCM,BaoSN,CampelloCCandFigueiredo
JR.ImpactofpituitaryFSHpurificationoninvitroearly
folliculogenesisingoats.Biocell:officialjournalofthe
SociedadesLatinoamericanasdeMicroscopiaElectronica
etal.2009;33:91-97.
5.MatosMH,Lima-VerdeIB,LuqueMC,MaiaJE,Jr.,
SilvaJR,CelestinoJJ,MartinsFS,BaoSN,LucciCM
andFigueiredoJR.Essentialroleoffolliclestimulating
hormoneinthemaintenanceofcaprinepreantralfollicle
viabilityinvitro.Zygote.2007;15:173-182.
6.CossD,JacobsSB,BenderCEandMellonPL.Anovel
AP-1siteiscriticalformaximalinductionofthefolliclestimulating
hormonebetagenebygonadotropin-releasing
hormone.TheJournalofbiologicalchemistry.2004;
279:152-162.
7.TelikicherlaD,AmbekarA,PalapettaSM,DwivediSB,
RajuR,SharmaJ,PrasadTsK,RamachandraY,Mohan
SS,MaharudraiahJ,MukherjeeSandPandeyA.A
comprehensivecuratedresourceforfolliclestimulating
hormonesignaling.BMCresearchnotes.2011;4:408.
8.GeorgeJW,DilleEAandHeckertLL.Currentconcepts
offollicle-stimulatinghormonereceptorgeneregulation.
Biologyofreproduction.2011;84:7-17.
9.KaiserUB,JakubowiakA,SteinbergerAandChinWW.
Differentialeffectsofgonadotropin-releasinghormone
(GnRH)pulsefrequencyongonadotropinsubunitand
GnRHreceptormessengerribonucleicacidlevelsinvitro.
Endocrinology.1997;138:1224-1231.
10.YeRS,XiQY,QiQ,ChengX,ChenT,LiH,KallonS,
ShuG,WangSB,JiangQYandZhangYL.Differentially
expressedmiRNAsafterGnRHtreatmentandtheirpotential
rolesinFSHregulationinporcineanteriorpituitarycell.
PloSone.2013;8:e57156.
11.DaiL,ZhaoZ,ZhaoR,XiaoS,JiangH,YueX,LiX,Gao
Y,LiuJandZhangJ.Effectsofnovelsinglenucleotide
polymorphismsoftheFSHbeta-subunitgeneonsemen
qualityandfertilityinbulls.Animalreproductionscience.
2009;114(1-3):14-22.
12.HeLandHannonGJ.MicroRNAs:smallRNAswithabig
roleingeneregulation.NaturereviewsGenetics.2004;
5:522-531.
13.ZhangB,WangQandPanX.MicroRNAsandtheir
regulatoryrolesinanimalsandplants.Journalofcellular
physiology.2007;210:279-289.
14.GuoH,IngoliaNT,WeissmanJSandBartelDP.
MammalianmicroRNAspredominantlyacttodecrease
targetmRNAlevels.Nature.2010;466:835-840.
15.LeeRC,FeinbaumRLandAmbrosV.TheC.elegans
heterochronicgenelin-4encodessmallRNAswithantisensecomplementaritytolin-14.Cell.1993;
75:843-854.
16.ReinhartBJ,SlackFJ,BassonM,PasquinelliAE,
BettingerJC,RougvieAE,HorvitzHRandRuvkunG.The
21-nucleotidelet-7RNAregulatesdevelopmentaltimingin
Caenorhabditiselegans.Nature.2000;403:901-906.
17.ZhangZ,FlorezS,Gutierrez-HartmannA,MartinJFand
AmendtBA.MicroRNAsregulatepituitarydevelopment,
andmicroRNA26bspecificallytargetslymphoidenhancer
factor1(Lef-1),whichmodulatespituitarytranscription
factor1(Pit-1)expression.TheJournalofbiological
chemistry.2010;285:34718-34728.
18.ElzeinSandGoodyerCG.Regulationofhumangrowth
hormonereceptorexpressionbymicroRNAs.Molecular
endocrinology.2014;28:1448-1459.
19.BhaskaranMandMohanM.MicroRNAs:history,
biogenesis,andtheirevolvingroleinanimaldevelopment
anddisease.Veterinarypathology.2014;51:759-774.
20.WienholdsEandPlasterkRH.MicroRNAfunctionin
animaldevelopment.FEBSletters.2005;579:5911-5922.
21.HoubaviyHB,MurrayMFandSharpPA.Embryonic
stemcell-specificMicroRNAs.Developmentalcell.2003;
5:351-358.
22.WienholdsE,KloostermanWP,MiskaE,Alvarez-Saavedra
E,BerezikovE,deBruijnE,HorvitzHR,KauppinenSand
PlasterkRH.MicroRNAexpressioninzebrafishembryonic
development.Science.2005;309:310-311.
23.BottoniA,ZatelliMC,FerracinM,TagliatiF,PiccinD,
VignaliC,CalinGA,NegriniM,CroceCMandDegli
UbertiEC.Identificationofdifferentiallyexpressed
microRNAsbymicroarray:apossibleroleformicroRNA
genesinpituitaryadenomas.Journalofcellularphysiology.
24.PalumboT,FauczFR,AzevedoM,XekoukiP,Iliopoulos
DandStratakisCA.Functionalscreenanalysisreveals
miR-26bandmiR-128ascentralregulatorsofpituitary
somatomammotrophictumorgrowththroughactivationof
thePTEN-AKTpathway.Oncogene.2013;32:1651-1659.
25.BottoniA,PiccinD,TagliatiF,LuchinA,ZatelliMCand
degliUbertiEC.miR-15aandmiR-16-1down-regulation
inpituitaryadenomas.Journalofcellularphysiology.2005;
204:280-285.
26.WeiZ,ZhouC,LiuM,YaoY,SunJ,XiaoJ,MaW,Zhu
HandWangR.MicroRNAinvolvementinametastatic
non-functioningpituitarycarcinoma.Pituitary.2015;
18:710-721.
27.SchneebergerM,AltirribaJ,GarciaA,EstebanY,Castano
C,Garcia-LavandeiraM,AlvarezCV,GomisRand
ClaretM.DeletionofmiRNAprocessingenzymeDicer
inPOMC-expressingcellsleadstopituitarydysfunction,
neurodegenerationanddevelopmentofobesity.Molecular
metabolism.2012;2:74-85.
28.ZhangN,LinJK,ChenJ,LiuXF,LiuJL,LuoHS,Li
YQandCuiS.MicroRNA375mediatesthesignalingpathwayofcorticotropin-releasingfactor(CRF)regulating
pro-opiomelanocortin(POMC)expressionbytargeting
mitogen-activatedproteinkinase8.TheJournalof
biologicalchemistry.2013;288:10361-10373.
29.NemotoT,ManoAandShibasakiT.Increasedexpression
ofmiR-325-3pbyurocortin2anditsinvolvementin
stress-inducedsuppressionofLHsecretioninratpituitary.
AmericanjournalofphysiologyEndocrinologyand
metabolism.2012;302:E781-787.
30.SheedyFJ.Turning21:InductionofmiR-21asaKey
SwitchintheInflammatoryResponse.Frontiersin
immunology.2015;6:19.
31.IorioMV,FerracinM,LiuCG,VeroneseA,SpizzoR,
SabbioniS,MagriE,PedrialiM,FabbriM,CampiglioM,
MenardS,PalazzoJP,RosenbergA,MusianiP,VoliniaS,
NenciI,etal.MicroRNAgeneexpressionderegulationin
humanbreastcancer.Cancerresearch.2005;65:7065-7070.
32.BandresE,CubedoE,AgirreX,MalumbresR,ZarateR,
RamirezN,AbajoA,NavarroA,MorenoI,MonzoMand
Garcia-FoncillasJ.IdentificationbyReal-timePCRof13
maturemicroRNAsdifferentiallyexpressedincolorectal
cancerandnon-tumoraltissues.Molecularcancer.2006;
5:29.
33.YanaiharaN,CaplenN,BowmanE,SeikeM,Kumamoto
K,YiM,StephensRM,OkamotoA,YokotaJ,Tanaka
T,CalinGA,LiuCG,CroceCMandHarrisCC.Unique
microRNAmolecularprofilesinlungcancerdiagnosisand
prognosis.Cancercell.2006;9:189-198.
34.ChaoTF,XiongHH,LiuW,ChenYandZhangJX.MiR-
21mediatestheradiationresistanceofglioblastomacells
byregulatingPDCD4andhMSH2.JournalofHuazhong
UniversityofScienceandTechnologyMedicalsciences=
HuazhongkejidaxuexuebaoYixueYingDewenban=
HuazhongkejidaxuexuebaoYixueYingdewenban.2013;
33:525-529.
35.BhagatTD,ZhouL,SokolL,KesselR,CaceresG,
GundaboluK,TamariR,GordonS,MantzarisI,Jodlowski
T,YuY,JingX,PolineniR,BhatiaK,PellagattiA,
BoultwoodJ,etal.miR-21mediateshematopoietic
suppressioninMDSbyactivatingTGF-betasignaling.
Blood.2013;121:2875-2881.
36.DasA,GaneshK,KhannaS,SenCKandRoyS.
Engulfmentofapoptoticcellsbymacrophages:aroleof
microRNA-21intheresolutionofwoundinflammation.
Journalofimmunology.2014;192:1120-1129.
37.AsanganiIA,RasheedSA,NikolovaDA,LeupoldJH,
ColburnNH,PostSandAllgayerH.MicroRNA-21(miR-
21)post-transcriptionallydownregulatestumorsuppressor
Pdcd4andstimulatesinvasion,intravasationandmetastasis
incolorectalcancer.Oncogene.2008;27:2128-2136.
38.BaiH,XuR,CaoZ,WeiDandWangC.Involvementof
miR-21inresistancetodaunorubicinbyregulatingPTEN
expressionintheleukaemiaK562cellline.FEBSletters.
2011;585:402-408.
39.SteelmanLS,NavolanicPM,SokoloskyML,Taylor
JR,LehmannBD,ChappellWH,AbramsSL,Wong
EW,StadelmanKM,TerrianDM,LeslieNR,Martelli
AM,StivalaF,LibraM,FranklinRAandMcCubreyJA.
SuppressionofPTENfunctionincreasesbreastcancer
chemotherapeuticdrugresistancewhileconferring
sensitivitytomTORinhibitors.Oncogene.2008;
27:4086-4095.
40.AmaralFC,TorresN,SaggioroF,NederL,MachadoHR,
SilvaWA,Jr.,MoreiraACandCastroM.MicroRNAs
differentiallyexpressedinACTH-secretingpituitarytumors.