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Promoter Hypermethylation of KiSS-1 Gene in Gastric Cancer

By Rosa Morales,2014-02-18 12:41
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Promoter Hypermethylation of KiSS-1 Gene in Gastric Cancerof,in,In,KiSS1,Gene,gene

    Promoter Hypermethylation of KiSS-1

    Gene in Gastric Cancer

    28DChinJCancerRes22(4):280284,2010www.springerlink.com

    PromoterHypermethylationofKiSS-1GeneinGastricCancer

    ZhiYang,DongQiuDai,YunYiDu

    DepartmentofSurgicalOncology,theFirstAffiliatedHospital,ChinaMedicalUniversity, Shenyang110001,China

    CLCnumber:R735.2Documentcode:AArticleID:1000-96O4(201O)O4-0280-05 DoI:10.1007/sll670.010.0280.8

    ChineseAntiCancerAssociationandSpringerVerlagBerlinHeidelberg20l0

    ABSTRACT

    Objective:Toinvestigatetheassociationbetween-1methylationandclinicopathologica1 characteristicsofgastriccancerandevaluatetheroleofperitoneallavagefluidindetectingperitoneal

    metastases.

    Methods:ThemethylationstatusofKiSS.1genein40gastriccancerspecimens,thecorresponding

    adjacentnormalmucosa,lymphnodesandperitoneallavagefluidwasinvestigatedbymethylationspcific

    polymerasechainreaction(MS.PCR1.

    Results:AberrantmethylationofKiSS

    1genewasdetectedin55%(22/401oftheadjacentnormal

    mucosa,82.5%(33/40)ofgastriccancerspecimens,80.95%(17/21)ofthelymphnodes,and42.5%(17/40)

    ofperitoneallavagefluid.Methylationingastriccarcinomaand1ymphonodewasmorefrequentthanin

    non

neoplasticgastiricmucosa.PresenceofKiSS.1methylationinperitoneallavagefluidwassig

    nificantly

    correlatedwithtumorinvasionrP=0.043).TheaccuracyofKiSS.1methylationinperitoneall

    avagefluid

    fordiagnosingperitonea1metastasiswas70%.withasensitivityof77.8%andaspecificityOf

    67.7%.

    Conclusion:AberrantmethylationofKiSS

    1geneisacommoneventintheoccurrenceandprogression ofgastriccarcinoma,whichmayprovideusefulinformationfortheearlydiagnosisofperitone

    almetastases

    andanewtherapyforgastriccancer.

    Keywords:Gastriccancer;Methylation;KiSS1;Peritoneallavagefluid

    INTRoDUCTIoN

    Gastriccanceristhesecondmostcommoncause

    ofcancerdeathintheworld11.Asitsmolecularbasis. deepinvolvementofaberrantDNAmethylationhas

    beenindicatedbythehigherincidencesofaberrant DNAmethylationofknowntumorsuppressorgenes

    thanofmutations.AberrantmethylationofDNA

    mayhappeninthepromoterCpGislandoftumor

    suppressorgenes.wheretranscriptionofDNAinto

    RNAbegins.Transcriptionisthefirstmaiorstepin decodingDNAintoaprotein.Animportantaspectof

    Received2010-01-20;Accepted2010-06-28

    ThisworkwassupportedbytheNationalNaturalScience FoundationofChina,(No.30572162);andtheScienceFoundation forHighEducationProgramofLiaoningProvince(No.2008S240) Correspondingauthor.

    Email:daidq63@163.corn

    themethylationmechanismisthatitinactivatestumor

    suppressorsgenes.Multiplerecentreviewshave shownthataberrantmethylationofDNAinpromoter CpGislandanddiminishedexpressionarepresentina numberoftumorrelatedgeneingastriccancer.For exampleRAssFlA.ancandidatetumorsuppressor gene,ishypermethylatedingastriccancer1TIMP3

    Whichhasalsobeensilenced,encodesaprotease inhibitorthatmayhelpinhibittissueinvasionL. KiSS.1anewlydiscoveredmetastasissuppressor gene.issilencedwithaberrantCpGisland

    hypermethylationingastriccancer?1.However,the

    relationshipbetweenmethylaitonofKiSs.1geneand gastriccancerhasnotbeenelucidatedyet. Inourstudy.KiSS.1genemethylationwas

    detectedinprimarytumor.thecorrespondingadjacent normalmucosa,metastaseslymphnodesand

    www.springerlink.comChinJCancerRes22(4):280284,2010281

    peritonealirrigationfluidusingmethylationspecial PCR.tOfindouttheassOciatiOnbetweentheKiSS,1 methylationandclinicopathOlOgica1characteristicsof gastriccancerandtodemonstratetheroleoftesting KiSS.1methylationofperitonealirrigationfluidin gastriccancerdiagnosis.

    MI'ERIALSANDMETHoDS

    CIjnicOpathOlogicalData

    Specimenswerecollectedfrom40caseswithits adjacentnormalmucosaofgastriccarcinoma undergoneasurgicalresectionintheSurgica1 OneologyDepartmentoftheFirstAffiliatedHospita1

ofChinaMedicalUniversityduringJuly2008'——

    January2009.Theenrolledpatientsincluded28males and12femaleswithameanageof56years(rang, 34.78years).A11ofthepatientsdidnotundergo chemotherapyorradiotherapybeforesurgical resection.

    Samples

    Fifrymlofpavsiologica1salinewereiniectedinto theDouglascavityatthebeginningoftheoperation andaspiratedaftergentlestirring,andthenthe peritoneallavagefluidwascollectedfromthecavity beforeasurgicalresection.Halfoftheperitoneal lavagefluidwasexaminedthroughconventional cytologicalmethodswithPapanicolaousstaining,and theremaininghalfwascentfifugedat2000r/min.for 20mintocollecttheintactcells.'andthenkeptina liquidnitrogentank.Thetissuesamplesincluding pnmarytumor,correspondingpaireda0jacentnormal mucosaandmetastaseslymphnodeswereprocured immediatelyafterresection.Sampleswerestoredat

    70.CuntilDNAextraction.A11thesamplesofthe primarygastriccancerwereevaluatedbytwo experiencedpathologistsfordiagnosis.Corresponding pairedadiacentnormalmucosawereobtainedatleast 3cmfromthedistalnegativesurgica1marginof40 patientsandtheabsenceofmalignancywasconfirmed histopathologically.Thelymphnodeswerestained withHEmethodtoconfirmwhetheratruemetastasis hadoccurred.Histologicalgradesandtumorcell

    differentiationwereconfirmedbyhistopathologica1 examinationandtheTNMstagewasaccordedtothe UnionInternationalContreleCancer(UICC. DNAExtractionandBisulfiteTreatment

    .

    70.Cuntiluse.TreatmentofDNAsampleswith bisulfiteconvertsallunmethylatedcytosinesto uracils.while1eavingmethylcytosinesunaltered.This allowsthesubsequentdifferentiationofmethylated andunmethylatedsequencesbymethylation.specific polymerasechainreaction(PCR).

    Methylation-specificPCRReaction

    DNAwaspurifiedusingaWizardDNAcleanup

    system(Promega)accordingtothemanipulation instrument.Thevolumeoftotalreactionmixturewas setat20pl,including3UlDNA,2pl10xPCRbuffer, 1.6B1dNTP,0.8LLlprimers(thesenseandantisense chain),0.15u1Taqenzyme,andl1.65laldouble. distilledwater.ThereactionconsistedofPre

    denaturingat94.Cfor5min,followedby35cycles, consistingof94.Cfor30s.58.Cfor30s.72.Cfor 20s.andwithafinalextensionat72.Cfor5min.The followingprimerswereusedforthedetectionof humanKisS1promotermethylation:Msense,5.

    AAAGTTTCGTTTCGGAGGGTTC.3'andManti.

    sense.5'.CTTTTATAAAACCCGAAATAACG3'for

    themethylatedsequenceofthehumanKiSS.1 promoter;Usense.5'.AAAGTTTTGTTTTGGAGG

    GTTT3'andUantisense.5'.AAAGTTTTGTTTT

    GGAGGGTTT.3'fortheunmethylatedsequenceof

    thehumanKsS.1promoter.Thepredictedproducts formethylatedandunmethylatedDNAwere172and 173bp.respectively.Theproductsweresubiectedto 2.5%agarosegelelectrophoresisat120Vfor40min andquantitatedwiththeFluorChen2.0system.DNA fromperipheral1ymphocytesofhealthYindividual andwaterwereusedasnegativecontrols.

    StatiscaiAnalysis

    Statisticalanalysiswasperformedusingthe SPSS13.0package.Chi.squaretestwasusedto analyzetherelationshipbetweenKiSS1methylation

    andclinicopathO1ogicalcharacteristiesinprimary tumoranditsmatchedadiacentnormolmucosa, metastaseslymphnodes.peritoneal1avagefluid. Fisher'sexacttestwasusedtoanalyzetheassociation betweenKiSS.1methylationinperitonea1lavagefluid andperitonealmetastasis.P<0.05wasregardedtobe statisticallysignificant.

    RESUI'S

    MethylationandCIinicopathoIocaICharac- teristics

    GenomeDNAwasextractedbythehydroxyl

    benzene

    chloroformextractionmethod,andstoredatTheKiSS-1methylationofthetUl/grosamplesa

    nd

    282ChinJCancerRes224):280284.2010www.springerlink,com itsmatchedadjacentnormalmucosa,metastases lymphnodeswasdetectedbymethylationspecia1.PCR (Figure1).Ourdatademonstratedthatmethylation wasfoundin82.5%(33/40)ofprimarytumor.in

    80.95%f17/21)ofmetastaseslymphnodesandin55% (22/40)ofmatchedadiacentnormalmucosa. 300-

    200?

    l00

    Methylationofingastriccarcinomaandlymphnode wasmorefrequentthaninnon?neoplasticgastiric mucosa,andthereweresignificantdifferences betweentheformertwoandthelatter.KiSS.1 methylationhadnosignificantrelationwiththe clinicopathologica1characteristics(Table1). UP123456

    /ViaMUMUMUMUMUMUMUMU

    Figure1.MSPresults:M:methylationresult;U:unmethylationresult;Ma:50bpDNALadder

    Marker;MP:methylation

    positivecontrol;UP:unmethylationpositivecontro1. Table1.TheassociationbetweenKiSS

    Jmethylationandclinicopathologicalcharacteristicsingastriccancer

    RelationshipbetweenPeritonealMetastasisand MethylationofPeritonealLavageFluid

    Inthestudy.weobservedthatthepromoterOf Kiss.1genewashyperrnethylated.ataratioof 42.5%(17,40).Amongthe19cases.9caseshad peritonealmetastasis.TheaccuracyotKiSS1 methylationinperitoneallavagefluidfordiagnosing peritonealmetastasiswas70%.withasensitivityof 77.8%,aspecificityOf67.7%,PPV41.2%,andNPV 91-3%(Table21.

    Table2.sociationbetweenKiSSJmeth),lationin

    peritobeallavageandperitonealmetastasis

PLMPeritonealmetastasis

    +

    PLM:methylationinperitoneallavage

    AsshowninTable3,thepresenceofKiSS.7

    methylationinperitoneallavagefluidwas significantlycorrelatedwithtumorinvasion www.springerlink.comChinJCancerRes22{4):280284,2010283

    (P=0.043).Theresultsoftheexperimentalsoshowed thatKiSSJmethylationinperitoneallavagefluidwas foundmorefrequentlyintumorwithlymphnode metastasisthanintumorwithoutlymphnode metastasis.

    Table3.AssociationbetweenKiSS1methylationin

    peritoneallavageuidandclinicopathologicafactors DISCUSSION

    Epigeneticalteration.conductedbytheDNA methyltransferases(DNMTs)catalyzingthe methylationofthe5positionofthecytosinering usingS.asenosylmethionineasthedonormoleculefor themethylgroup.hasbeenshowntoplayan

    importantroleintumorigenesisandprogression【引.

    MethylationofpromoterCPGislandsleadsto transcriptiona1silencingoftheirdownstreamgenes. Invarioushumancancers,silencingoftumor suppressorgenes,suchasCDKN2AIp16),CDHl (Ecadherin)andMLH1.isknowntobeoneofthe maiormechanismsfortheirinactivation,alongwith mutationsandL0H.KiSS1isametastasis-

    suppressinggenefoundinhumanmelanomacelllines in1996andpeptidesderivedfromitsproductare

naturalligandsoforphanproteincoupledreceptor

    GPR54.LotsofresearcheshavesuggestedthatKiSS1

    geneisassociatedcloselywithmetastasesof melanoma[1o].

    breastcancer[,2],

    thyroidcancer[13,

    esophagealcancer[14.

    gastriccancer[15andpancreatic

    cancer

    .Themechanismsofthedecreased

    expressionofKiSS.1appeartobemultifactorial,and morestudiesneedtobeelucidated.Toexporethe novelmethylation.silencedgenesingastriccancer, Yamashitaetalcarriedoutachemicalgenomic

    screening,agenomewidesearchforgenesup

    regulatedbytreatmentwithademethylatingagent, 5aza2deoxvcvtidine(5azadC).After5azadC

    treatmentofagastriccancercellline(AGS)579 geneswereupregulated16fl0ldormore.usingan

    oligonuc1eOtidemicroarraywith39000genes.The KiSS1wasupregulated54.8.fold.anditsmethylation wasfoundin80%(8/10,ofprimarygastriccancers. Thisresultisapproximatestoourresults. Inthisstudy.westudiedtheassociationbetween KiSs1methylationandprimarytumor.corresponding pairedadjacentnormalmucosaandmetastases1ymph nodesingastriccancer.Theresultsofthisexperiment indicatethatthemethlytioninprimarytumor(82.5%1 andmetastaseslymphnodes(8o.95%,wasmuch higherthancorrespondingpairedadjacentnormal

    mucosa(55%1.andthereweresignificantdifierences betweentheformertwoandthelatter,respectively. KiSs1methylationwasnotcorrelatedwiththe clinicopathologicalcharacteristics.

    Methylationalterationcanbefoundnotonlyin solidcancertissuesbutalsoinvariOUSremote samplesderivedfrompatientswithcancer,suchas sputumforlungcancerL'.urineforurologic tumors.salivaforheadandnecksquamouscell carcinoma[.

    breastfluid[21andserumorplasmafor

    almostalltypesofcancer【一….Morerecentstudies

    havebeenca~iedouttodetectdifferencesinvarious remotesamplesanddemonstratedthatDNA

    methylationcouldbeactasapromisingbiomarkerin earlydetectionandprognosis.Inourstudy.the associationbetweenKiSS.1methylationinperitoneal 1avagefluidandperitonealmetastasiswasexamined. eobservedthatthepromoterofKiss1genewas

    hypermethylatedataratioof42.5%(17/40).The presenceofKiSS1methylationinperitonea1lavage fluidwassignificantlycorrelatedwithtumorinvasion fP=0.043).Thereasonmaybethattheincidenceof freecancercellsfa11offintoperitonealcavity increaseaccordingtothedepthoftumorinvasion. Amongthe17caseswithpromoterhypermethylation. 9caseshadperitonealmetastasisincluding cytologicallypositiveperitoneallavage.KiSS.1 promoterhypermethylationinperitoneallavagefluid showedahighersensitivity(77.8%,forthediagnosis

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