Similar Squamous Cell Carcinoma Epithelium microRNA Expression in Never Smokers and Ever ...

16 Pages·2015·1.27 MB·English

RESEARCHARTICLE
Similar Squamous Cell Carcinoma Epithelium
microRNA Expression in Never Smokers and
Ever Smokers
AntoniaKolokythas1,YaluZhou2,JoelL.Schwartz2,GuyR.Adami3*
1 DepartmentofOralandMaxillofacialSurgery,CollegeofDentistry,UniversityofIllinoisatChicago,801
SouthPaulinaStreet,Chicago,Illinois,60610,UnitedStatesofAmerica,2 ArphionLtd,2242W.Harrison
Street,Chicago,Illinois,60612,UnitedStatesofAmerica,3 DepartmentofOralMedicineandOral
Diagnostics,CenterforMolecularBiologyofOralDiseases,UniversityofIllinoisatChicago,801South
PaulinaStreet,Chicago,Illinois,60610,UnitedStatesofAmerica
*[email protected]
Abstract
Theincidenceoforaltumorsinpatientswhoneverusedmutagenicagentssuchastobacco
OPENACCESS
isincreasing.InanefforttobetterunderstandthesetumorswestudiedmicroRNA(miRNA)
Citation:KolokythasA,ZhouY,SchwartzJL,Adami
expressionintumorepitheliumofnevertobaccousers,tumorepitheliumofevertobacco
GR(2015)SimilarSquamousCellCarcinoma
EpitheliummicroRNAExpressioninNeverSmokers users,andnonpathologicalcontroloralepithelium.Acomparisonoflevelsamong372miR-
andEverSmokers.PLoSONE10(11):e0141695. NAsin12nevertobaccouserswithoralsquamouscellcarcinoma(OSCC)versus10
doi:10.1371/journal.pone.0141695
healthycontrolswasmadeusingthereversetranscriptionquantitativepolymerasechain
Editor:Pei-YiChu,SchoolofMedicine,FuJen reaction.Asimilaranalysiswasdonewith8evertobaccouserswithOSCC.Thesecompari-
CatholicUniversity,TAIWAN
sonsrevealedmiR-10b-5p,miR-196a-5p,andmiR-31-5pasenrichedinthetumorepithe-
Received:July28,2015 liuminOSCCofbothneverandevertobaccousers.ExaminationofTheCancerGenome
Accepted:October11,2015 Atlas(TCGA)projectmiRNAdataon305OSCCsand30controlsrevealed100%ofthose
miRNAsenrichedinneversmokerOSCCsinthispatientgroupwerealsoenrichedinever
Published:November6,2015
smokerOSCCs.NonsupervisedclusteringofTCGAOSCCswassuggestiveoftwoorfour
Copyright:©2015Kolokythasetal.Thisisanopen
subgroupsoftumorsbasedonmiRNAlevelswithlimitedevidencefordifferencesin
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits tobaccoexposureamongthegroups.Resultsfrombothpatientgroupstogetherstressthe
unrestricteduse,distribution,andreproductioninany importanceofmiR196a-5pinOSCCmalignancyinbothneverandeversmokers,and
medium,providedtheoriginalauthorandsourceare
emphasizetheoverallsimilarityofmiRNAexpressioninOSCCsinthesetworiskgroups.It
credited.
impliesthattheremaybegreatsimilarityinetiologyofOSCCinneverandeversmokers
DataAvailabilityStatement:Allrelevantdataare
andthatclassifyingOSCCbasedontobaccoexposuremaynotbehelpfulintheclinic.
withinthepaperanditsSupportingInformationfiles.
Addtionaldataareavailableathttps://tcga-data.nci.
nih.gov/tcga/.
Funding:Thefunderprovidedsupportintheformof
salaryforauthor[Y.Z.],butdidnothaveany
additionalroleinthestudydesign,datacollectionand Introduction
analysis,decisiontopublish,orpreparationofthe
manuscript.Thespecificroleofthisauthoris MicroRNAs(miRNAs)inmatureformarenoncodingRNAs,19to25nucleotidesinlength,
articulatedinthe‘authorcontributions’section. withtheabilitytoinhibitthetranslationandshortenthehalf-lifeofmRNAs[1].MiRNAscan
directlyregulatemultiplemRNAs,whichencodetheproteinsthatcontrolimportantcellular
CompetingInterests:Y.Z.andJ.S.areaffiliated
withArphionLtd.acompanythatseekstodevelop processes.Manyoftheseregulatedpathways,includingapoptosis,cellproliferation,andcell
PLOSONE|DOI:10.1371/journal.pone.0141695 November6,2015 1/16
MicroRNAExpressionSimilarinSmokerandNonsmokerOralCancer
cancertreatmentsanddiagnostics.Thismanuscript migration,canalsocontributetocancer[2–4].Thereareover2000knownmiRNAs,asubset
doesnotrelatetoanyproductorproductin ofwhichhavebeenshowntoshowchangesinlevelsthatcorrelatewithvariouscancers[5,6]
developmentatArphionLtd.Thisrelationshipwiththe
(http://mirbase.org/).GlobalexpressionanalysisofthesemiRNAsindifferentcancershas
sponsordoesnotaltertheauthors'adherenceto
identifiedmiRNAsthatfunctionasoncomirs,likemiR-21-5p,andareconsistentlyupregulated
PLOSONEpoliciesonsharingdataandmaterials.
insomecancertypes,whileothermiRNAsarereducedincertaintumortypesandappeartobe
tumorsuppressors[7].Varioustumortypeshavebeencharacterizedtoshowasignatureof
miRNAlevelsassociatedwiththesetumorsandtheirprogression,whichmayaidindiagnosis
andprognosis[5,6].ThesmallsizeandregulatoryfunctionofmiRNAshavealsomadethem
thefocusofresearchusingthemorsimilarmoleculestochangetumorcellpropertiesandthus
treatcancer[8–10].
MuchefforthasgoneintodescribingasetofmiRNAsthatshowconsistentchangesinlev-
els,firstwithheadandnecksquamouscellcarcinomas(HNSCCs)[11–13]andmorerecently
withsubsetsofthesecancerssuchasoralsquamouscellcarcinoma(OSCC)[14].Theresultsof
theseanalyseshaveshownsomeconsistencies,suchasfairlyuniversalupregulationofmiR-21-
5p,andsomewhatlowerconsensusonotherpotentialoncomirs,probablyduetothevariable
amountofmixedepithelium/stromainsamplesanddiversityofetiologyofthetumorsubtypes.
Forexample,oralpharyngealcancer,unlikeOSCCeveninnonsmokers,isoftenassociatedeti-
ologicallywithtransformingHPV,specificallyHPV16[15–17].Recentworkhasbroughtto
lighttwodistinctetiologiesofOSCC,thoseassociatedwiththemainriskfactorknown,tobacco
usage,andthosenot[18–20].Likesmall-celllungcancerinneversmokers,OSCCsinnever
tobaccousersseemtobedistinct[14].Hereafter,inthisreport,wewillabbreviatethisgroup
whodonotusetobacco,orothermutagenicproductssuchasbetelnut,to“neversmokers”.
OSCCinneversmokers,whichisontheincreaseintheUnitedStates,seemstostrikeonaver-
agebotholderandyoungerpatientsthanthoseassociatedwithtobaccousage.Italsotendsto
presentinearlierstage,andoccursmostfrequentlyinthetongueandgingivanotthefloor-of-
mouthwheretobacco-associatedOSCCsoccur.Molecularly,OSCCsinneversmokersshow
lowerratesofp53genemutations,andthereissomeevidenceofdifferencesingeneexpression
[19–22].Liketobacco-relatedOSCCstheyarerarelyassociatedwithtransformingHPVor
enrichmentofthep16tumorsuppressor[23–26].Fewerthan10%oforalcancersareHPV
geneexpressionpositiveeveninpatientswithnohistoryoftobaccouse[24,26].Overalllittleis
knownabouttheetiologyorthechangesinmRNAormiRNAassociatedwiththissubtypeof
OSCC[24]
Wequantifiedlevelsof372miRNAsin12OSCCepithelialsamplesfromneversmokers
versus10samplesfromacontrolgroupofsubjectswithapparentlynormaloralmucosa.We
alsotestedlevelsofthesemiRNAsinatestgroupofOSCCsassociatedwithtobaccousage.
Next,wedidsimilarcomparisonsusingthemiRNAexpressiondataofthe344controland
OSCCsamplesfromTheCancerGenomeAtlas(TGCA)HNSCCcohort.Tumorsamplesof
thisstudyweredissectedsurgicallyandcontainsomestromaltissue.Togetherweusedthese
datasetstocomparemiRNAexpressioninneverandeversmokerswiththegoalofstartingto
gaininsightonetiologyofOSCCinthesetwodifferentOSCCriskgroups.
MaterialsandMethods
Clinicalsampling
Twobrushcytologysampleseachwerecollectedfrom12subjectswhoneverusedtobacco,or
othermutagenicagentslikebetelnut,andwhopresentedwithorallesionsthatwerebiopsy-
provenOSCC.ThesepatientswereseenintheOralandMaxillofacialSurgeryClinicinthe
UniversityofIllinoisMedicalCenter.Samplesfromnormalcontrolswerefrom10never
tobaccousersfromoralsitesthatwerenormalonclinicalexaminationbytheoralsurgeon.The
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MicroRNAExpressionSimilarinSmokerandNonsmokerOralCancer
secondgroupofbrushcytologysampleswastakenfrom18currentorformertobaccousersat
lesionsitesofeitherOSCCornonmalignantdiseasewithintactmucosa.Benignsamples
includedmucosallesionssuchasleukoplakia,allwithoutdysplasia.Alldiagnoseswereverified
byhistopathologicexaminationofsurgicallyobtainedtumortissueforOSCCsandscalpel
biopsymaterialfornon-malignantlesions.Allsubjectsinallgroupsprovidedwrittenconsent
toparticipateinaccordancewithguidelinesoftheOfficefortheProtectionofResearchSub-
jectsoftheUniversityofIllinoisatChicago,thelocalInstitutionalReviewBoardthatformally
approvedofthisresearch.
Brushcytology
Brushcytologywasperformedonpatientsastheypresentedintheclinicjustpriortobiopsyas
describedearlier,takingcaretosampleareaswithintactepithelium[27].Sampleswereimme-
diatelyplacedinTrizol(LifeTechnologies,Carlsbad,CA,USA),mixed,andfrozen.Weuseda
cervicalcytologybrushwithRNApurificationasdescribedinSchwartzetal.[27].
RNA
RecentpublicationshavestressedtheproblemswithusageofTrizoltoisolatemiRNAwitheth-
anolorisopropanolprecipitationswhenRNAlevelsshowawiderange[28].Whilecellswere
storedinTrizol,weusedamethodologysimilartothatrecommendedbyKimetal.andimme-
diatelyfollowingphaseseparationallsamplesweresubjectedtosilicate-basedbindingpurifica-
tiontopreventselectivemiRNAloss(S1Fig).WeusedRNeasychromatography(Qiagen,
Germantown,MD,USA)toremovemRNAfollowedbyethanoladditionandRNeasyMinE-
lutechromatography(Qiagen)tobindthenelutesmallRNAs,includingmaturemiRNA.
Therewasa6-foldrangeinsampleRNAlevelsbasedonRT-PCRwithsimilaraveragelevelsin
themalignantandnonmalignantgroups.
QuantitativeRT-PCR
10ngRNAwasreversetranscribedin5ulreactionsusingthemiRCURYLNAUniversalRT
microRNAPCR,PolyadenylationandcDNAsynthesiskit(Exiqon,Woburn,MA,USA).
cDNAwasdiluted20foldandassayedin10ulPCRreactionsaccordingtotheprotocolfor
miRCURYLNAUniversalRTmicroRNAPCRagainstapanelof4miRNAsandaspike-in
controlforcDNAsynthesis.Ofeachsamplepairfromasinglesubject,thesamplewiththe
higheryieldbasedonreversetranscriptionquantitativepolymerasechainreaction(RT-qPCR)
wassubjectedtoascaledupcDNAsynthesisandwasassayedoncebyRT-qPCRonthemicro-
RNAReady-to-UsePCR,HumanpanelI(Exiqon),whichincludes372miRNAprimersets.
Negativecontrols,excludingtemplatefromthereversetranscriptionreaction,weretestedand
profiledlikethesampleswithindividualprimerpairs.Theamplificationwasperformedinan
AppliedBiosystemsViia7RT-qPCRSystem(LifeTechnologies,Carlsbad,CA,USA)in384
wellplates.TheamplificationcurveswereanalyzedforCtvaluesusingthebuilt-insoftware,
withasinglebaselineandthresholdsetmanuallyforeachplate.ResultswithmiRNAsshown
tobedifferentiallyexpressedintheinitialscreenwerecorroboratedusingasimilarRT-PCR
assayminimallyinduplicate(Exiqon).
miRNAdataanalysis
ForRT-qPCRdataanalysis,40miRNAswereselectedasstandardstonormalizeCtvaluesfor
eachplate.ThesereferenceswerechosenbecausetheywereamongalargesubgroupofmiRNAs
expressedinallsamples.WeusedthedeltadeltaCtmethodtocalculateexpressionvalues.All
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MicroRNAExpressionSimilarinSmokerandNonsmokerOralCancer
CtvalueswereimportedintotheRankProductprogram,whichrankslevelsforeachmiRNA
withinasample,multipliesthevaluesforallsamplesinonegrouptogettherankproduct,and
thencalculatesacombinedprobabilityofthedistributionforeachRNAinthetwogroupsto
determinetheprobabilityofdifferentialexpression[29,30].Acutoffforthepercentage(pro-
portion)offalsepositivesof0.05istakenassignificantfordifferentialexpression.TCGARNA-
seqdatafor314OSCCsamplesand30controls,weredownloadedformtheTCGADataPortal
(https://tcga-data.nci.nih.gov/tcga)asnormalizedmiRNAquantificationfilesalongwithaccom-
panyingpatientclinicalinformationfiles.TheexactnamesforTCGA-derivedmiRNAswere
obtainedbyexaminingasubsetof5individualsampleisoformquantificationfilestoidentify
eachdifferentiallyexpressedmiRNAbasedonitsmappedgenomicsiteasthe3por5pisoform.
Whenambiguousthisdesignationwasnotgiven.NormalizedmiRNAlevelcountswereloaded
directlyintotheRankProditProgramtoperformrankproductsanalysis[29,30].Nonnegative
MatrixFactorizationConsensusClusteringwasusedtoidentifytheoptimalnumberofdistinct
samplesclustersamongthe305OSCCsintheTCGAdatasetwithknownsmokingstatus
accessedthroughtheGenePatternportalwww.broadinstitute.org/.Itwasusedtoidentifythe
optimalnumberofdistinctsamplesclustersamongthe305OSCCsintheTCGAdataset[31–
33].First,allmiRNAswithmorethan50%sampleswithzerovalueswerefilteredout.Clustering
wasthendonebasedonthelevelsofthe238miRNAswhichshowedthegreatestvariationin
expressionlevels(normalizedstandarddeviation>1).Thecopheneticcoefficientderivedserved
asameasureofcorrelationbetweenthesampledistanceinducedbytheconsensusmatrix.
HeatmapsforvisualpresentationofthemiRNAexpressiondataweregeneratedusingBRB
Arraytools[34].FortherepresentationofRNAfrombrushcytologydatasetweusedhierar-
chicalclusteringwith1-correlationandaveragelinkageoftheexpressionlevelsof50miRNAs
showntobedifferentiallyexpressedbetweenOSCCandnonOSCCsbasedonclasscomparison
usingBRBArraytools.FortherepresentationofTCGAexpressiondatanonnegativematrix
factorizationwasusedtoclustersamplesbasedontheexpressionlevelsof228mostvariably
expressedmiRNAs.
Results
miRNAexpressioninOSCCineversmokers
ThisworkusesRNAfromoralmucosalcellsobtainedbybrushcytology[27,35]sowefirst
soughttoverifythisapproachtomeasuremiRNAlevelsbyexaminingmiRNAsassociated
withOSCCintobaccousers.Tofocusonmalignancy-specificpathologicchanges,wecom-
paredexpressionofmiRNAsinOSCCsofeversmokersversusthatinnonmalignantlesionsin
asimilarpopulation.WecomparedepithelialmiRNAfromOSCClesionsin8patients,and
nonmalignantorallesions/conditionsof9tobaccousers,asoutlinedinTable1.Theseincluded
agranularcelltumor,mucosalaberrationssuchasfibroushyperplasiaandhyperkeratosis,and
asofttissueameloblastomaofthegingiva.Theselesionsoftenshowincreasedcellproliferation
andpossiblyinflammation,butnototherpropertiessuchasblockstoapoptosisandthe
increasedtissueinvasionthatcanoccurwithmalignancy.Weusedtherankproductmethodol-
ogy,anonparametricstatisticaltool,todeterminedifferentiallyexpressedmiRNAs[29,30].
ThistestformiRNAdifferentialexpressionwithOSCCineversmokersrevealedonemiRNA
thatwasinducedspecificallywithOSCCusingthecriteriaof>2-foldchangeandatp<0.05
fortherankproducttest(Table2A).ThisinducedmiRNAwasmiR-196a-5p,amiRNAassoci-
atedwithoralcancerinmanystudies.NomiRNAsshowedadecreaseinexpression.
Manypublishedstudiesonoralcancerlargelyfocusonbetel-ortobacco-associatedcancers
andcompareRNAintumorsversusnormalnonpathologicaltissue.WealsocomparedmiR-
NAsenrichedwithtobaccoassociatedOSCCversusmiRNAsinnormaloraltissue.Withthis
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MicroRNAExpressionSimilarinSmokerandNonsmokerOralCancer
Table1. EverSmokerPatientdata.
Sample Sitea Sex Age Tobacco/Betelb Ethanolc Path.Stage Graded ImmuneState
OSCC110 T M 50 Bet+Tob None T2N0M0 WellDiff Norm
OSCC129 LG M 52 F-Tob L/M T1N0M0 WellDiff Norm
OSCC329 LG M 59 F-Tob None T4aN0M0 PoorlyDiff Norm
OSCC359 T M 61 Tob Heavy T4N2cM0 ModDiff Norm
OSCC416 T M 61 Tob L/M TcisN0M0 N/A Norm
OSCC449 LG F 62 Tob None T4NoM0 ModDiff Norm
OSCC466 T M 64 Tob Heavy T2N0M0 PoorlyDiff HIVpositive
OSCC485 FOM M 56 Tob None T4aN2bM0 ModDiff Norm
Sample Site Sex Age Tobacco/Betel Ethanol Path.Finding ImmuneState
BL117 LM F 46 Tob L/M CanicularAdenoma Norm
BL129 Bu M 27 Tob None Ameloblastoma Norm
BL149 LG F 54 F-Tob None Hyperkeratosis Norm
BL319 T F 33 Tob L/M GranularCellTumor Norm
BL367 Bu M 44 ST None Hyperkeratosis,Mucositis Norm
BL474 LG F 77 F-Tob L/M Fibroushyperplasia Norm
BL482 T M 58 Tob L/M Hyperkeratosis Norm
BL490 UG M 51 Tob L/M FibrousHyperplasia Norm
BL495 UG M 60 Tob None Hyperkeratosis Norm
aT,tongue;LG/UGlower/uppergingiva;FOM,floorofmouth;Bu,buccalmucosa,LM,lipmucosa.
bTob,Tobaccouser;ifformeruserthanF-Tob.;Bet,beteluse;ST,smokelesstobaccouser
cL/M,lighttomoderateintakeofethanolupto50gramsperday,Heavy,intakeofethanol>50g/day.
dwelldifferentiated,WellDiff;moderatelydifferentiated,ModDiff;Poorlydifferentiated,PoorlyDiff
doi:10.1371/journal.pone.0141695.t001
approachwesawinductionof8miRNAsincludingmiR196a-5p,miR-10b-5p,miR-31-5p,
miR-451aandmiR-144-3p(Table2B).Ofthese,besidesmiR-196a-5p,miR-10b-5pandmiR-
31-5phavebeenshowntobeenrichedinearlierstudiesofheadandneckcancerusingsurgi-
callyobtainedwholemucosa,andmiR144-5pandmiR-451awereshowntobeinducedinthe
salivaofthosewithheadandneckcancer[36–38].
miRNAexpressioninOSCCinneversmokers
NeversmokerpatientfeaturesaresummarizedinTable3.Therewere12patients,7females
and5males,withagesrangingfrom37to90years,withtheaverageageof67.Thecontrol
groupofsubjectsrangedinagefrom28to77;withanaverageof61.Therewere6femalesand
fourmales.MiRNAlevelsintheseneversmokerOSCCswerecomparedtothatinnonpatholo-
gicmucosainneversmokers.
Among372miRNAsanalyzedseven,miR-196a-5p,miR-10b-5p,miR-503-5p,miR-451a,
miR-144-3p,miR-187-3pandmiR-31-5p,showedincreasedexpressionintheOSCCsamples
oftheneversmokers,againusingtherankproductmethodology(Table2c)[29,30].Fig1
showsaheatmapwithrelativeexpressionofthedifferentmiRNAsinsamplesfromever
smokerOSCC,neversmokerOSCC,andneversmokercontrols.Again,nomiRNAsshowed
downregulation.Thismaybebecausethetumorsamplesareoftenamixtureoftumorand
normalepitheliumwhilecontrolsamplesarepurenormalepitheliummakingalossofexpres-
sionintumorhardtodetect.
ConfirmationofsimilarOSSCmiRNAexpressionchangesineversmokersandnever
smokers. TheTCGAdatasetofglobalmiRNAexpressiondataof305OSCCandcontrol
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MicroRNAExpressionSimilarinSmokerandNonsmokerOralCancer
Table2. MicroRNAsenrichedinOSCCandbenignorallesions.
A.EversmokerOSCCversuseversmokerbenignpathology
MicroRNA FoldinductionwithOSCC PValueb Pfpb
miR-196a-5p 8.7 0.000104 0.008
miR-144-3pa 7.9 0.000104 0.01
B.EversmokerOSCCversusneversmokernormal
MicroRNA FoldinductionwithOSCC PValue Pfp
miR-196a-5p 61.0 0.000 0.000
miR-144-3pa 103.9 0.000 0.000
miR-451aa 92.9 0.000 0.000
miR-10b-5p 32.8 7.81E-05 0.006
miR-31-5p 16.1 0.000234 0.015
C.NeversmokerOSCCversusneversmokernormal
MicroRNA FoldinductionwithOSCC PValue Pfp
miR-196a-5p 19.2 2.60E-05 0.005
miR-10b-5p 15.0 2.60E-05 0.010
miR-503-5p 15.0 0.000156 0.012
miR-451aa 11.7 0.000391 0.025
miR-144-3pa 9.6 0.000859 0.033
miR-187-3p 10.0 0.000859 0.037
miR-31-5p 6.7 0.001250 0.040
amicroRNAhighlyexpressedinblood,
brankproductprobability
cpfpisfortherankproducttest:percentagefalsepositivepredictions
doi:10.1371/journal.pone.0141695.t002
Table3. NeverSmokerOSCCPatientdata.
Sample Sitea Sex Age Ethanolb PathologicStage Gradec ImmuneState
OSCC231 T M 55 None T1N0M0 ModDiff Norm.
OSCC305 T M 78 L/M T1N0M0 ModDiff Norm.
OSCC308 T F 74 L/M T1N0M0 ModDiff RheumatoiArthritisd
OSCC3553 LG F 84 None T1N0M0 ModDiff Norm.
OSCC357 T F 74 None T4N0M0 ModDiff Norm.
OSCC413 T F 63 None T3N0M0 ModDiff Norm.
OSCC453 Bu F 37 L/M T2N0M0 ModDiff Norm.
OSCC463 LG F 69 None T2N0M0 ModDiff Norm.
OSCC4231 LG M 65 None T4N0M0 ModDiff Renaltransplante
OSCC4281 LG F 58 None T2N0M0 ModDiff Norm.
OSCC4291 T M 90 None T3N0M0 ModDiff Norm.
OSCC5271 LG M 54 None T4N0M0 ModDiff Renaltransplante
aT,tongue;LG,lowergingiva;Bu,buccalmucosa.
bL/M,lighttomoderateintakeofethanolupto50gramsperday,Heavy,intakeofethanol>50g/day.
cwelldifferentiated,WellDiff;moderatelydifferentiated,ModDiff;Poorlydifferentiated,PoorlyDiff
dtreatedwithanti-inflammatoryandTumorNecrosisFactorinhibitor,etanercept
etreatedwithstandardpost-transplantimmunosuppressivemedications
doi:10.1371/journal.pone.0141695.t003
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MicroRNAExpressionSimilarinSmokerandNonsmokerOralCancer
Fig1.DifferentiallyexpressedmiRNAsexpressedincontrolsandOSCCsfromneversmokersandeversmokers.Supervizedhierarchicalclustering
of50differentiallyexpressedmiRNAswithfoldchangesabove2,among9controls,12neversmokerOSCCs,and8eversmokerOSCCs.Epithelialsamples
arelistedinthecolumnsandmiRNAsareinrows.Redindicateshigherexpressionthanaverageandblueindicateslowerthanaverage.
doi:10.1371/journal.pone.0141695.g001
mucosasamplesallpreparedunderstandardmethodslinkedtoclinicaldatawasusedtofurther
exploremiRNAsineversmokersversusneversmokers.Thesetconfirmedtheobservationthat
eversmokersandneversmokersOSCCsshowsimilarmiRNAlevels.Weperformedclasscom-
parisonamongthe217eversmokerOSCCsamplesversusthe20eversmokercontrols
(Table4a,S5Table).Wethendidthesamebetweenthe88neversmokerOSCCsamplesversus
the10neversmokercontrols(Table4b,S6Table).All10miRNAsenrichedintheneversmoker
OSCCsbyrankproductwerecontainedinthelistofmiRNAsenrichedwiththeeversmoker
group.TheeversmokergroupshowedmoremiRNAsdifferentiallyexpressedbutthatmaybe
duetothelargersizeoftheeversmokerdatasets.NomiRNAsshowedreducedlevelsinthe
neversmokerstumorthoughthreeweredepressedintheeversmokertumorgroupversuscon-
trol,miR-375,miR-1-2-3p,andmiR-99a(Table4c).Whenadirectcomparisonbetweenever
smokerandneversmokerOSCCmiRNAexpressiondatawasdonewesawonlyonemiRNA
wasdifferentiallyexpressed,miR-637,confirminghowsimilarthetwogroupsare(Table4d).
ItwasshownsometimeagothatHNSCCfallinto4subtypesbasedonglobalmiRNA
expression,thoughoneofthegroupsisheavilyweightedtooralpharyngealtumors[39–41].
Weperformedunsupervisedclusteringusingnonnegativematrixfactorization(NMF),which
identifiescommongene/RNAexpressionpatterns,ormetagenes,amongsamples[31].Itcalcu-
latesacopheneticcoefficientforeachvalueofK(numberofclusters)whichismaximalwhen
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MicroRNAExpressionSimilarinSmokerandNonsmokerOralCancer
Table4. miRNAsdifferentiallyexpressedinOSCCandcontrolsinTCGAdataset.
A.EversmokerOSCCversuseversmokernormal
MicroRNA FoldinductionwithOSCC PValuea Pfpb
miR-196b-5p 16.5 0 0
miR-503-5p 8.0 0 0
miR-196a-5p 21.0 0 0
miR-1293 12.1 0 0
miR-196a-5p 10.9 0 0
miR-937-3p 8.5 0 0
miR-210-3p 7.2 0 0
miR-31 11.1 0 0
miR-105-2 35.4 0 0
miR-1269a 52.7 0 0
miR-767 29.3 0 0
miR455 6.2 9.56E-06 0.000714
miR-615-3p 6.7 9.56E-06 0.000769
miR-105-1 25.9 9.56E-06 0.000833
miR-548f 14.0 0.000153 0.010667
miR-1910 7.8 0.000315 0.020625
miR-3648 4.7 0.000478 0.029412
miR-4326 4.9 0.000602 0.035000
miR-224 4.8 0.000746 0.041053
B.NeversmokerOSCCversusneversmokernormal
MicroRNA FoldinductionwithOSCC PValue Pfp
miR-105-1 75.8 0 0
miR-105-2 87.3 0 0
miR-196a-5p 11.1 0 0
miR-196b-5p 16.4 0 0
miR-210-3p 5.1 0 0
miR-503-5p 9.2 0 0
miR-767 69.5 3.82E-05 0.00571
miR-31 4.9 4.78E-05 0.00625
miR-1269a 14.5 5.73E-05 0.00667
miR-455 6.7 0.000421 0.04400
C.DepressedineversmokerOSCCversuseversmokernormal
MicroRNA FoldinductionwithNormal PValue Pfp
miR-375 0.085 0 0
miR-1-3p 0.14 6.69E-05 0.0175
miR-99a 0.23 0.000182 0.038
D.EversmokerOSCCversusneversmokerOSCC
MicroRNA Foldinductionwitheversmokers PValue Pfp
miR-637 3.4 0 0
arankproductprobability
bpfpisfortherankproducttest:percentagefalsepositivepredictions
doi:10.1371/journal.pone.0141695.t004
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MicroRNAExpressionSimilarinSmokerandNonsmokerOralCancer
Fig2.DiscoveryofOSCCsubtypesbasedonmiRNAexpressionamong305OSCCsintheTCGAdataset.A)Nonnegatiivematrixfactorization
consensusclusteringshowedsupportfortwoandfourOSCCsubtypesbasedonthemaximalcopheneticvalueatK=2and4.B)Consensusmatricesfor
thesampleswerecalculatedatk=2,3,4,5,6,7,8.Acomparisonofthevisualizedconsensusmatricesallowedavisualassessmentofthebestrank.Clear
blockpatternsalongthediagonaloftheconsensusmatricesindicatedrobustnessoftheclustering.C)Analysisoftheeversmokersandneversmokersin
bothmiRNAbasedsubclassrevealedalackofadifferenceintheproportionsofthetworiskgroupsbasedonsmokingexperience,p<0.059byFisherExact
TestD)Analysisoftheeversmokersandneversmokersineachof4miRNAbasedsubclassesrevealednosignificantdifferenceinproportionsofeverand
neversmokersinanygroup.
doi:10.1371/journal.pone.0141695.g002
theclustersshowmaximalseparation.Usingthe238miRNAsmostvariablyexpressedamong
the305OSCCsamplesK=2–7producedthehighestcopheneticcoefficientatK=2and4
indicatingtwoorfourclustersofsamples(Fig2)[32].Aheatmapreveasdifferentialexpres-
sionofasubsetofthemiRNAsusedtoseparatethecasesintotwosubtypesorclusters(S2Fig).
GiventhesetwoOSCCgroups,weexaminedtobaccousageamongthesubjectsandfoundit
waspossibleonegroupshowedaslightlyhighernumberofsmokersbutthisdidnotreach
PLOSONE|DOI:10.1371/journal.pone.0141695 November6,2015 9/16
MicroRNAExpressionSimilarinSmokerandNonsmokerOralCancer
significance(Fig2C).Whenwecomparedpackyearexposurebetweenthesetwogroupswe
foundthefirstgroupshowedalmost40%lowerexposure626¼2.6packyearsversusthesecond
100+4.4witht<0.036.SuggestingtobaccoexposuremayindeedhavesomeeffectonmiRNA
expression.
When4OSCCsubclasseswereexaminedfortobaccousagetherewasnosignificantdiffer-
enceinproportionofeverandneversmokersbasedontheChiSquaretestthoughsomegroups
weresmallmakingstatisticalanalysisdifficult(Fig2Aand2D).
Discussion
TheetiologyofOSCCofneversmokersisunknown.IfOSCCinneversmokersisadistinctsub-
typeofthiscancerthenregulatoryRNAsinthetumorepitheliummaybedifferentthanthoseof
tobacco-associatedOSCCs.Tumorsfrombothcohortsaregenerallynotassociatedwithp16
enrichmentortransformingHPVgeneexpressionandthatwasthecaseherewithonesubject
positiveforp16andonepositiveforHPV16RNA(S2Table)[24,26].Thisstudysoughtto
examineOSCCepitheliumtodiscernmiRNAmisexpressionwiththisdiseaseinneversmokers.
WemeasuredmiRNAsenrichedinOSCCepitheliumofneversmokersandineversmokers
bothcomparedtonormaltissue.WefoundthatmiR-144-3p,miR-451a,miR-10b-5p,mir-31-
5p,andmiR-196a-5pwereenrichedwithtumorformationwhetherthesubjectwasanever
smokerorneversmoker.ExaminationoftheTCGAOSCCsandmiRNAexpressionalsosug-
geststhatoveralltherewasmuchalikeinOSCCsfromeverandneversmokers(S3Table).
ItwaspossibletobaccoplayedaroleinchangingmiRNAexpressioninasubgroupof
OSCCs;howeverithasbeennotedthatthe“classical”subtypeofHNSCCs,associatedwith
heavytobaccousage,isonlyaminorcomponentofOSCCs.IndeedaspublishedtheTCGA
groupshowedonly11.2%ofgroupof178oraltumorsshowedthe“classical”mRNAexpres-
sionprofile[39].WhenweseparatedtheOSCCsbasedonmiRNAexpressionwefoundtwoor
foursubclasses(Fig2).Thetwosubclassclustersshowedthemaximalagreementwiththe
mRNAbasedsubclassificationswithgrouponehavingmostofthe“atypical”mRNAexpres-
sionpatternandgrouptwohavingmostofthe“classicalpattern”[40,41].Curiouslygrouptwo
ofthetwosubclassesshowedanominallyhigherpercentageofsmokersthatdidnotreachsta-
tisticalsignificance,buttherewasa40%increaseincigarettepackyearexposure62±2.6versus
100±4.4,t<0.0389.ThesefindingsshowtobaccousagemayhavesomeeffectonmiRNA
expression,butotherunknownfactorshavelargereffects,andthatrelyingontobaccoexposure
toassigntumortypeisnotwise.
Thisstudyinpartfocusesononecelltype,epithelium,obtainedbybrushcytology,making
itmoresensitivetochangesinexpressionofmiRNAsfoundmainlyinthisspecificcelltype
[27].MiRNAsenrichedinstromaandnotepithlieumoftumorswouldnotbeapparent.Itis
importanttonotethatanythingthatcausesthebrushtoacquirecellsnormallynotpresentin
theepithelium,suchastheepithelialinvasionoflymphocytesthatcanoccurininflamed
OSCC,canalsoresultinchangesinRNAsinthesample.Somesamplesmayalsohaveblood
contaminationwhileothersdonot.Inparticular,malignantlesionscanbemorehighlyvascu-
larizedwithbloodvesselsnexttoepithelialcells,whichcangreatlyincreasethemixofblood
cellsexposedtothebrush.WesawelevatedlevelsofmiR-451ainbothtumorgroupscompared
tonormaltissue.ThisRNAishighlyexpressedinredbloodcells(RBCs)andisawell-known
indicatorofRBCRNAcontaminationinplasmasamples[42,43].MiR-451alevelswerehigh-
estinOSCCsamplesofeversmokersandlowestinnormalsamplesofneversmokers.Inthe
samples,levelsofmiR-451acorrelatedwithasecondblood-linkedmiRNA,miR-144-3p,witha
correlationcoefficientof0.91[36,37,44],datanotshown.WhileweshowedbothmiR-451a
andmiR-144-3paremarkersforOSCC,thispropertyisalmostcertainlyindirectandthereis
PLOSONE|DOI:10.1371/journal.pone.0141695 November6,2015 10/16