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Inhibition of RAW264.7 Macrophage Inflammatory Cytokines Release by Small Haparin RNAi Targeting TLR4

By Julia Pierce,2014-02-18 10:38
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Inhibition of RAW264.7 Macrophage Inflammatory Cytokines Release by Small Haparin RNAi Targeting TLR4

    Inhibition of RAW264.7 Macrophage Inflammatory Cytokines Release by Small

    Haparin RNAi Targeting TLR4

    JournalofHuazhongUniversityofScienceandTechnology[MedSci]

    华中科技大学[医学(英德文)]26(5):500503,2006

    InhibitionofRAW264.7MacrophageInflammatoryCytokinesRe-

    leasebySmallHaparinRNAiTargetingTLR4.

    WANGHui(王慧),ZHANGJinxiang(张进祥),wuHeshui(吴河

    ),JIANGChunfang(蒋春舫),

    ZHENGQichang(郑启昌),LIZhuoya(李卓娅)

    lDepartmentofMedicalBiology,3DepartmentofImmunology,TongfiMedicalCollege,HuazhongUniversityofScienceaddTech_

    nology,Wkhan430030,China

    2Denm.tmentofSurgery,UnionHospita1.TongjiMedicalCollege.HuazhongUniversityofScienceandTechnology,Wuhan430022

    China

    Summary:Inordertoconstructanexpressionvectorcarryingsmallhairpin(sh)RNA(shRNA)fl0r

    toll

    likereceptor4mRNAandareportergeneofenhancedgreenfluorescenceprotein(EGFP)and studytheinhibitionofcytokinereleasebyRAW264.7cellinducedbylipopolysaccharide(LPS)

    stimulationthroughtransfectionandexpressionofshRNAtargetingTLR4geneviatheRNAimecha

    nism.thereportergeneplasmidpEGFP.Cl(4.7kb)andpsiRNA_hHlneo(2979bp)wereused.The

    HlpromotoranddoubleBbsIrestrictendoenzymesitewereclonedfromplasmidpsiRNA-hHlneo

    andreconstructedthemintoplasmidpEGFP

    ClintheMlulrestrictendoenzymicsite,forming

    plasmidPEGFP.Hl/siRNA.whichcontainedBbssiteandreporterEGFPgene.Thenanoligonuclear

    hairpinsequencetargetingTLR4genewasdesignedbyinternettoolandinsertedintotheplasmid

    pEGFPHl/siRNAformingplasmidpEGFPHl/TLR4siRNA.Aftertransfectionof

    pEGFP.Hl,]rLR4siRNAintoR_AW264.7cells,tumornecrosisfactoralpha(TNF

    a)releasebythe

    cellsafterstimulationbvLPSwasdetected.Theresultsshowedthattheconstructed pEGFP

    H1,]ruR4.siRNAcarryinghairpinRNAforTU4geneandreporterEGFPgenewereproven toberightbyrestrictionendonucleaseanalysis.TheexpressionofEGFPgenewas(5O.37_+8.23)%

    andaftertransfectionoftheplasmidpEGFPHl,TLR4siRNAthelevelofTNF

    releasedby

    RAW264.7eellwasdownregulated.ItwasconcludedthatshRNAtargetingTLR4genecouldinhibit

    theTNFareleasebyRAW264.7cellsevokedbyLPS.

    Keywords:RNAinterference;lipOpOlysaccharide;macrophage;tolllikereceptor4

    DOIl0.1007/sl1596006-0503x

    RNAinterferencefRNAi)isakindofnewlydevel

    opedgenesilencingtechnique.Genespecificsup

    pressinginmarnmalianeelIscanbeachievedbydeliver

    ingofsmallinterferingRNAs(siRNAs)thatare2123

    nucleotideinlength.longenoughtointroduce

    genespecificsuppression,butshortenoughtoevadethe

    hostinterferonresponse.siRNAcanbeproducedbysuch

methodsaschemicalsynthesis,invitrotranscript,deg

    radationoflongfragmentdouble.strandRNAbvRNase III.siRNAexpressingvector.Amongthemthemethod ofexpressingvectoristhemostconvenientandtheex_ pressionofsiRNAcanbekeptinalongperiod.

    Tol1.1ikereceptor4fTLR4)recognizeslipopoly- saccharide(LPS,releasedbygramnegativecellwalls. AftertherecognitionTLR4recruitsaseriesofsignal moleculesthroughitscytoplasticTollrIRmotifand

    activatesnuclearfactor.kappaB(NF.KB),resultingin thedownstreaminflammatorycytokinestormcharacter

    izedbyreleaseofpro.inflammatorycytokinesasTNF.0L Becauseofthecomplexityofinflammatorycytokine WANGHui,female,bornin1977,M.D.,Ph.D.

    Correspondingauthor

    ThisprojectwassupportedbygrantsfromNationalNatural SciencesFoundationofChina(No.30200272andNo. 30500487).

    networkandthetwowayeflfectofcytokines.whichwere identifiedbynumerousresearchesdirectingtothemiddle signalmolecules,asNFrd3orterminalcytokines,as

    TNF0Lnoidealresultswereharvesteduptonowunder theconditiontheresearcheswerejustcarriedonevents fromthedownstreamofLPSstimulationI''.Sowetry tostudythemacrophageinflammationstimulatedby LPSfromtheveryoriginofthemembranereceptorby methodofRNAitechnique.

    AplasmidpEGFP-C1.whichcarriesenhanced

    greenfluorescenceprotein(EGFP),wasselectedtocon.

structanexpressionvectorpEGFP.H1rLI4.RN.

    whichcarriedshRNAforTLR4mRNAbyinsertionof humanHlpromotorandshRNAholdingsites.Theinhi. bitionofcytokinereleasebvRAW264.7cellsstimulated byLPSthrou2htransfectionoftheplasmidandthere. suitingexpressionofshRNAtargetingTLR4genevia theRNAimechanismwerestudied.Wehopetolaya foundationfortheapplicationofRNAibyexpressing vectorpatterningeneexpressionregulation. 1MATEIuALSANDMETHoDS

    1.1ConstructionofRNAieukaryoticexpressing vectorofpEGFP-H1/siRNA

    ThevectorofpsiRNAhHlneo(InvivogenCo.,

50l

    LA1ncltidedHlpiomotnranddoubleBbslrestric

    tlOllellzvmeCUtlnSites.tileJalterwasIorclorlealltl expressionof0nheparinRNA(shRNA1.Bvmeth(1d ofpolymerasechainJeactioninwhichupairofprimers carryingM2"1enz\,mecuttingsiteWeredesigned.,?:

    duplicatedafragmenf'rol1]psIRNAhHlneewhichin

    cludedthettIprolltOtOla11ddoubleBb1reslr1cte1] euzymecUttjngsitesttheLenhwas500bp)Andthi

    fi-agmentWaSC1onedintoa11otherplasmidpEGFPC1

    fDepartmentoihnmullnlogyTol1iMcdjcIl_Colege,

    HSTChtl&aMluI(MBICo.USAlrestrictionen

    zymecuttingsltetbrmi1ganewenkarvotLcshRNAex-

    pressingplasmidpEGFPHJ/RNAThentheneW

    plasmidwasdentlfledbyBbslfMBlCo.USA)re

strictj('ellzyulecutandsequencnganaysiS.

    1.2ConstructionofaRNAjPlasmid

    pEGFP-HlrIR4.RNATargetingTLR4mRNA

    DirectedbvhcslRAdesIgnsoftwal'efRNA

    Wizard.".Invlv(cnCoUSA)apairofpalindrc)111e sequenceswiththelengthel55hntargetiniJ.TLR4 mRNAfNM(12l2971wasdeslened.w]lIchbegnwith

    file1159thbasefl'Olnthestcodon.Thesequellcesre: 5TCCCGGTTGCTGrrClrrA1(_rGA1CAAGAGATC AGAATAAGAACAGCAACC1TT3and5CAAAAAaG

    1rCrGn'CTA]rrCTGATl'CTTGAATCAGAATAAGA ACAGCAACC3:CX)lltlolsequt2nucs:5'TCCCGTGTA CTAl:TClrrAC?ICTGA几几(AAGAGATCAGA6TAAaAGT

    AGTACAC兀一3and5CAAAAAGlGTACTACTCTT

    A(1CTGAT1?AAGAGATCAGAGTAAGAGrAGTACAC3

    onh)th5overhangtrod3overhangltesBIrestricton

    enzymecuttingsitewasinsertedThestalkstructure%aS TCAAGAGThellesefragllielllwcrensertedillto

    pEGFP-Hl,RNAatBb1restrictionellzvmecull1n sIteformingafle',,vplasrl|idpEGFP-HI,lR4RNA

    midltsconireIpEGFPHl/contrO]S1RNABothnew

    plasmidswerejdentcdhvluJrestr1cli011eDzV111e._ cutandsequenclllganalysis

    1.3Culture(IfRAW264.7CelIIJinn

    sfDepar111ellt ML1finemaclophageRAW2647ceI

    ofImmttuology,TonedjcaCoIleg.e.HUST.Cbina

    wereresuscitatedSki,;pendedandeuIluredn164()ih 0%FCSjna5%COalmospherelit37?.fheme

    diumwaschangedevery24h

    1.4LipofectamineMediatedpEGFP.H1/

    TLR4.siRNArransfeetionandIdentification Directedbythema11Uatheplasmid

    pEGFPHI,1_LR4siRNAwasuonlposedi11tOl_pofec

    tamine2O0(j(InvitrogellCoUSA1andtransfectc~the

    ptasmid11tOR_AW264.7cellsAner24htheexpresso11 0fEGFPjnthecellsWasobservedthrouehfluorescence 1nvertedmicroscopecExcitalionwaveIeuglh488nm.

    EmIssionwavelength507nln1toCValuatetheeffectof" transfectionThecellsweredigestedwIth025%trvDsiu. 11owedby'1tRR)r/minr0r5111inThecellswerewashed t0r2timesbyPBS.theconcentrationofcellsvcasad

    justedtoI)<l0<'/mLandtheexpressionofGFPwas

    fu~hcranalyzedbytlowcylontetry.

    1.5LevelsofTNF?tlinSupernatan!afterLPS StimIllationtoJAW264.7

    Cellswasdividedinto:TLR4-RNAl(thecells transfiectedwithpEGFP-H1/TLR4siRNA)group,COO

    Ire]aNAlgroup(cellstransfectedwith

    pEGFPHI/controlsiRNAlandshamcontrolgroup

    fcellstransfeetedwiihblanklipofectamine)Thesuper- nata1]teleachoupwascollectedrespectivelyat2h mid8haffel10tltgfLLPSstimulationandkeptat--70 '_rheIeveIsofTNF-wereana1yzedby'nlethodof ELISAusdirectedbythelllunual

    I.6StatislieAnalvsis

    AIlthenumericdatawerelndieatedarhDq

    werealla1yzedbvSPSS0)wjthaStudenc?ftest

    2RESULTS

    2.1IdentificationofEukaryoticRNAiExpressing

VenlorofPEGFPH1/stRNA

    AfterBIrestrictionenzvn]eCUtandeeutropho-

    resisonl5皤一a._osegeItilereconstructPlasntidcarry

    li1gEGFPw1ththe11antepEGFPHl,sIRNAwasCOF

    recttfig1)Andthesequc_]cina11aJysjsaIsovenlied thecorrect

    Fig.2pEGFP-HI/TLR4Mlurestrictenmccul MMarker:IshRNA2:CentreI

    2.3EvaluationofEGFPExpressionofIL-%W264.7 Cells

    Underthesan]ecellconcentrationtheexpressionof

502

    EGFPincellstrans[ectedwithpEGFPItI/TLR4-rsiRNA

    orpEGFPHI/conlroIsiRNAindicatedtheidentical ~II{)re$cencedensityunder1"~uorescencein~ertedo3tcro

    scopecfig.3)Fh)wcytometryshowedthepercenrageol, EGFPpn,sitivecellwas{5037_+823

    2.4Levelut'TNF-"(pmLlinCultureSuper. natantofRAW264.7CellsstimulatedbvLI CelIsinRNAigroupshowedhighresistanc~toLPS stimulationaindicatedbythedownI'eguIatcdTNF一?

    levelscomparedwithIhalinRNAicontrol01.shamcon

    trolgroupsffig4).

    Fig.3AftertransfectionofEGFPHIll'LR4siRNAandpEGFP-HI/'conlrolsiRNAlnIlRAW2~7mutingmat'rophagecellline thevariationofEGFPe~pression

    A.B:x2(}0CD'x44)0

    500

    400

    300

    Z

    200

    Slimulati'1Il2h

    Nhnlulati'11I1

    :t.广

    Fig.4InhibitionolcylokinereleaseolRAW2647byP,hRNA reedlnedRNAJ

    Comparedwithcontrolg~roup.,,<(n01p<c)Ol 3DISCUSSION

    RNAiisaconservedbologicresponsetOdou

    ble?strandedRNAfdsRNA1thatresultslnthese

    qnencespeciticsilencingttargetgeneexpressionIn recenlyearsthemechanismunder1yingRNA1wasestab- lishedfrOlIlmuchworkondiverseorj~ansmsasthe

    "

    ,e\+ornlanduit1]yAswellasbeingnvo1red1nbait?ng

    viruses.RNAijsprobablYifflportantjnmaintainjnorder mnegenomebysuppresslngthemovemenIe1]lloblle geneticIemetssuchastranspasonsandlepelitB.ese

    quences..TheRNAmachiner~mavalsohavearolein

    finetuningnormalcellulargeneexpression ln,.,estigationoftheRNAjmachineryhasre\:ea1ed thermatinuandcentralroleel'slRNA.speciesol,2I nucleotidesincngththatarcper1)ctlvcoulpelllentarvto

    thetargetRNAAsequencewasconstrucledthatcnn

    tainedan_n;,erredrepeatofafragmentofthetalgelgene sequence,separatedbyaspacerthatWLtSanllitton.and encodedahairpinRNAARertrgnsl'ormati(yllthehairpin

    producedfromthevectoljshilvefficIentnknock1ng Oownexpresslonc)1thetargetgene.AsaresLiltasVslenl t0rIhestableexpressOIlofSiRNAshasbeendeveloped nWillcnrnapnmallanexpresslotlveclol'weredes1gned t0directintraceIIularSVDesIsofsiRNAsInnloslcases.

    thetargetspecificjnserlimadeupofal9i1LIC1eelide

    sequencenon]Pelllentiffvothetarget.I】【)w.dbya

    sh<}rtspacerLindthere\.erseCOUlp]enleulofthesame targetsequenceOncetranscrjbed.al9bpstem一】OOp

    SLrLICtLII'e.1ermedshRNA.iprocessedbyDicerjntoa sjRNAthatcandiJeelthedownretlIatIonoftargetene

    expressionx.iaIhee]enlentsOIRNAjnlach_'cryllI.

    Busedontheseresealcheswecnnsll-uctedanew RNAivectolcpEGFPHl,RNA)bCUlllnHlpro

    motorandthedc'wI1streanlset.Itlellcebetweeubothbs

    lrestrictionenzyn1e[esonlpsIRNA-HIliceand ree1)nstruttedthemlntopEGFPCl?Mhr1restricti(1.11

    enzymesleF['lIOWIngSRNAWjzardelltheintemet thup://www.simawi:.'.ard0In/)wedesi1ledLmdsynthesize apairelspeclicsequeuCCNconIaIningulooptargeting BlurjngTLR4mRNAandIlcolnpIementandinserted ch1ssequenccjnlopEGFPHlIRNAtlt61restfic

    lionellz}qllecultingsltelOI_ecoqlstrut[anewshRNA expressingvectorofpEGFp-H1rLR4.sjRNABecause

    elarepal'1geneEGFPonthisplasnlldweCOUIdmoutot

    theefficacY'rranshwmafionhvdetectndensjtvof

    i"luorescence.AtierIrans(ormallof1urtherct:onstructed plasmid1ntoRAW2647.theefticacvnrexpl'essjonf1f EGFPWaSkeptalfileIeveI()r50-(fig31).Sowecould

    discrjmihatecel_sexpressingshRNAbs'detcotnextent

offluorescencenrtdel11UOFCSCCnCCins,'enedmUloscope

    andfacjlitatingthe-lna1ysiselsenesi1enciuhvvectors exp]essingshRNA

    T("l一】ikereceptorsareakindofI1ew1vl"OUndtrans

    membranereceplors,whjchalelcratedc,nthesLirfilceof macrophagesTheiraclivaton1sthecritIcaIjnitintinn

    pointpr~e,dingthelnf]antlt]atoryI_eactionInducedby endotoxinandexotoxjn.,a;hichaeproducedby~raln negativeandgranlpositivebarter1a.A】】illemberseltile

    (0lIram1lysharesimlIarextracellulardomainsincluding 183IeucinerichrepealsfLRRs}..midsireliarCVtO

    plasmicdomainsofapproxinlateY20()1t[11inoaeids.

    whichareaIsosimilartothecytoplasmicdomain')fthe lL-Irece13Vor{theTIR

    l'egion)17lTLR4isone01-

    illenlhersoftollfitmi15'It

    filL1receptorlhomologous

    themostclearlyidentified

    recognizesLPSindttcinga

    seriesc~oplaslicproteininteraction.wh1chjn"lr1re.

    suts?1prIIouf_dprnnammmtvcv1ok1llerclclse suchasTNF_Whcnerci门【Iexece0rfh【卜

JournalofHuazhongUniversityofScienceandTechnology[MedSci]26(5):2006503

    gens.TLI4couldrecognizedangersignalsreleasedby damagedcellsortissues.Asakeypoint.TLR4initi-

    atesinnateimmunesystemandtriggersadaptiveimmune response,inducingtheinflammatorycas

    

    cad

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