Duan Z_2009_J Orthop Res
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DifferentialExpressionofmicroRNA(miRNA)inChordomaRevealsaRoleformiRNA-1inMetExpression
ZhenfengDuan,1EdwinChoy,1G.PeturNielsen,2AndrewRosenberg,2JohnIafrate,2CaoYang,1JoeSchwab,1HenryMankin,1RamnikXavier,3FrancisJ.Hornicek1
1CenterforSarcomaandConnectiveTissueOncology,SarcomaMolecularBiologyLaboratory,MassachusettsGeneralHospital,50FruitStreet,
Jackson1115,Boston,Massachusetts02114,2DepartmentofPathology,MassachusettsGeneralHospital,Boston,Massachusetts,3CenterforComputationalandIntegrativeBiology,MassachusettsGeneralHospital,Boston,Massachusetts
Received28July2009;accepted20October2009PublishedonlineinWileyInterScience(www.interscience.wiley.com).DOI10.1002/jor.21055
ABSTRACT:EmergingevidencesuggeststhatmicroRNA(miRNA)expressionsignaturesincancermayhaveimportantdiagnostic,prognostic,andtherapeuticvalue,butthereisnodataonmiRNAexpressioninchordoma.ThepurposeofthisstudywastoidentifytheroleofmiRNAsinhumanchordoma.WeanalyzedmiRNAexpressioninchordoma-derivedcelllinesandchordomatissuebyusingmiRNAmicroarraytechnologywithunsupervisedhierarchicalclusteringanalysis.TherelativeexpressionlevelsofthesemiRNAswereconfirmedbyreal-timequantitativeRT-PCRandNorthernblotanalysis.TocharacterizethepotentialroleofmiRNA-1,miRNA-1wasstablytransfectedintoachordomacellline,UCH1.TheexpressionofmiRNA-1targetedgeneMetinchordomatissueswasalsostudied.WeobservethathumanchordomatissuesandcelllinescanbedistinguishedfromnormalmuscletissuebycomparingmiRNAexpressionprofiles.SeveralmiRNAsweredifferentiallyexpressedinchordomacelllinescomparedtocontrols,andsimilarexpressionpatternswerefoundinprimarychordomatissues.Importantly,wewereabletoshowforthefirsttime,toourknowledge,thatexpressionofmiRNA-1andmiRNA-206,twomiRNAsimplicatedinanumberofothercancertypes,weremarkedlydecreasedinbothchordomatissuesandcelllines.WhenchordomacelllinesweretransfectedwithmiRNA-1,downregulationofknownmiRNA-1targetswasobserved.ThesetargetsincludedMetandHDAC4—twogenesthatwereobservedtobeoverexpressedinchordoma.OurresultsdemonstratethatsomemiRNAsaredifferentiallyexpressedinchordomaand,inparticular,miRNA-1mayhaveafunctionaleffectonchordomatumorpathogenesis.ß2009OrthopaedicResearchSociety.PublishedbyWileyPeriodicals,Inc.JOrthopRes
Keywords:chordoma;miRNA;Met;RT-PCR
Chordomaisanunusualraretumorofbonethatusuallyarisesintheaxialskeletonalongthetractofthenotochord;themostcommonlocationisthesacrumwhere50%ofthesetumorsdevelop.1Traditional
treatment,surgicalresectionandradiation,isoftendifficultbecauseoftheneedtopreserveadjacentvitalstructures.Accordingly,completeremovaloftumorisoftennotpossibleandthisisassociatedwithlocalrecurrenceratesofover40%insomeseries.2,3Addition-ally,therearenoeffectivesystemictherapiesavailable.Therapeuticadvancesareneededtoimproveoutcome,andnewfocusisbeingdirectedatidentifyingimportantmolecularpathwaysamenabletotargetedtherapy.MicroRNAs(miRNAs)aresmall,noncoding,single-strandedsegmentsofendogenousRNAscontaining19–25nucleotides(nt)inlengththatcontrolgeneexpressionandfunctionbydownregulatingmessengerRNA(mRNA)expression.4ThegenesthatencodemiRNAsaretranscribedfromDNAbutnottranslatedintoprotein.miRNAsareprocessedfromprimarytranscriptsknownaspre-miRNAtoformshortstem-loopstructurescalledprecursormiRNA(pre-miRNA).5,6Thepre-miRNAissubsequentlyconvertedtofunctionalmaturemiRNAinthecytoplasm;onceinthecytoplasm,miRNAsarecleavedfurtherbytheenzymeDICERwhichresultsinthecreationofmiRNAsthatareassociatedwithacellularcomplexthatissimilartotheRNA-inducedsilencingcomplexthatparticipatesinRNAinterference.Itisbelievedthatthereareasmanyas1,000miRNAsinthehumangenome,andupto30%ofhumangenesareregulatedbymiRNAs.ElucidatingthefunctionofmiRNAintumorpathogenesisandprogressionisimportantastheymayplaycriticalrolesintheregulationofgenesinvolvedinthecontrolofdevelop-ment,cellproliferation/differentiation,apoptosis,anddrugresistance.Finally,severalstudieshavefoundmiRNAprofilesareusefulintherefinementofdiagnosisandprognosisofhumancancers.7–9ThestatusofmiRNA
expressioninchordomashasnotyetbeenassessed.ThegoalofthisprojectistostudymiRNAexpressionprofilesinhumanchordoma.
MATERIALSANDMETHODSHumanChordomaTissueandCellLinesSpecimensofprimarychordomawereobtainedduringthesurgicalremovalofpreviouslydiagnosedtumorsandwereusedinaccordancewiththepoliciesoftheinstitutionalreviewboardofthehospital.Alldiagnoseswereconfirmedbylightmicroscopyandimmunohistochemistry.TheUCH1celllineisanestablishedchordomacelllineandwasgenerouslysuppliedtoourgroupbyDr.SilkeBruderlein(UniversityHospitalsofUlm,Germany).UCH1wasestab-lishedfromarecurrenceofasacralchordomaandisthefirstconfirmedhumanchordomacellline.10UCH1exhibits