Effects of arecoline on proliferation of oral squamous cell carcinoma cells by dysregulating c-Myc

16 Pages·2017·5.85 MB·English

RESEARCHARTICLE
Effects of arecoline on proliferation of oral
squamous cell carcinoma cells by
dysregulating c-Myc and miR-22, directly
targeting oncostatin M
JureepornChuerduangphui1,2,TipayaEkalaksananan1,2,PonlathamChaiyarit3,4,
NatchaPatarapadungkit2,5,ApinyaChotiyano2,6,BunkerdKongyingyoes7,
SupanneePromthet2,8,9,ChamsaiPientong1,2*
a1111111111
a1111111111 1 DepartmentofMicrobiology,FacultyofMedicine,KhonKaenUniversity,KhonKaen,Thailand,2 HPV&
EBVandCarcinogenesisResearchGroup,KhonKaenUniversity,KhonKaen,Thailand,3 Departmentof
a1111111111
OralDiagnosis,FacultyofDentistry,KhonKaenUniversity,KhonKaen,Thailand,4 ResearchGroupof
a1111111111
ChronicInflammatoryOralDiseasesandSystemicDiseasesAssociatedwithOralHealth,Facultyof
a1111111111 Dentistry,KhonKaenUniversity,KhonKaen,Thailand,5 DepartmentofPathology,FacultyofMedicine,
KhonKaenUniversity,KhonKaen,Thailand,6 AnatomicalPathologyUnit,KhonKaenHospital,KhonKaen,
Thailand,7 DepartmentofPharmacology,FacultyofMedicine,KhonKaenUniversity,KhonKaen,Thailand,
8 DepartmentofEpidemiology,FacultyofPublicHealth,KhonKaenUniversity,KhonKaen,Thailand,
9 ASEANCancerEpidemiologyandPreventionResearchGroup,KhonKaenUniversity,KhonKaen,
Thailand
OPENACCESS
*[email protected]
Citation:ChuerduangphuiJ,EkalaksanananT,
ChaiyaritP,PatarapadungkitN,ChotiyanoA,
KongyingyoesB,etal.(2018)Effectsofarecoline
onproliferationoforalsquamouscellcarcinoma Abstract
cellsbydysregulatingc-MycandmiR-22,directly
targetingoncostatinM.PLoSONE13(1): Arecoline,themajoralkaloidofarecanut,isknowntoinduceoralcarcinogenesis,however,
e0192009.https://doi.org/10.1371/journal.
itsmechanismisstillneededtoelucidate.Thisstudyinvestigatedtheeffectsofarecolineon
pone.0192009
cellviabilityandcell-cycleprogressionoforalsquamouscellcarcinoma(OSCC)cellsas
Editor:AamirAhmad,UniversityofSouthAlabama
wellasarelevantcellulargeneexpression.Theresultsshowedthatalowconcentrationof
MitchellCancerInstitute,UNITEDSTATES
arecoline(0.025μg/ml)increasedOSCCcellviability,proportionofcellsinG2/Mphaseand
Received:October12,2017
cellproliferation.Simultaneously,itinducedIL-6,STAT3andc-Mycexpression.Interest-
Accepted:January15,2018 ingly,c-mycpromoteractivitywasalsoinducedbyarecoline.MiR-22expressioninareco-
Published:January31,2018 line-treatedOSCCcellswassuppressedandcomparabletoanupregulatedc-Myc
expression.Inarecoline-treatedOSCCcells,oncostatinM(OSM)expressionwassignifi-
Copyright:©2018Chuerduangphuietal.Thisisan
openaccessarticledistributedunderthetermsof cantlyupregulatedandinverselycorrelatedwithmiR-22expression.Likewise,OSMexpres-
theCreativeCommonsAttributionLicense,which sionanditspost-transcriptionalactivityweresignificantlydecreasedinmiR-22-transfected
permitsunrestricteduse,distribution,and
OSCCand293FTcells.ThisresultdemonstratedthatmiR-22directlytargetedOSM.Inter-
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. estingly,miR-22playedanimportantroleasatumorsuppresseronsuppressingcellprolifer-
ation,migrationandcell-cycleprogressionofOSCCcells.Thisresultsuggestedtheeffect
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation ofarecolinetopromotecellproliferationandcell-cycleprogressionofOSCCcellsmightbe
files. involvedininductionofc-MycexpressionandreductionofmiR-22resultinginOSM
Funding:ThisstudywasgrantedbyKhonKaen upregulation.
University,Thailand(GrantNumbers592002and
601504)andreceivedscholarshipunderthePost-
DoctoralTrainingProgramfromResearchAffairs
andGraduateSchool,KhonKaenUniversity,
Thailand(Grantno.58443).
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Dysregulatingc-MycandmiR-22,directlytargetingoncostatinMbyarecolineinOSCCcells
Competinginterests:Theauthorshavedeclared Introduction
thatnocompetinginterestsexist.
ArecanutchewingthatismostfrequentlydoneinAsia,isamajorriskfactorfororalsqua-
Abbreviations:293FT,Humanembryonickidney
mouscellcarcinoma(OSCC)[1].Arecolineisthemainalkaloidinarecanutandisknownto
293FTcellline;EMT,Epithelial-mesenchymal
havecytotoxic,genotoxicandmutagenicproperties,contributingtohistologicchangesand
transition;FBS,Fetalbovineserum;miR,
otherbiologicalconsequences[2,3].Itislikelythattheeffectsofarecolinevarydependingon
microRNA;MTT,3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazoliumbromide;Mut,Mutant;OSCC, celltype,individualidiosyncrasyanddose.However,littleisknownasyetaboutthevarious
Oralsquamouscellcarcinoma;OSM,Oncostatin effectsofarecoline.
M;PBS,Phosphate-bufferedsaline;RT-PCR,Real Activationofc-Mycisacriticalprocessincancerdevelopment/progression[4].Various
timepolymerasechainreaction;UTR,Untranslated
factorscaninducec-Mycexpressionbyactivationofmitogenicsignalingcascades,including
region;WT,Wildtype.
IL-6/STAT3signalingcascade,etc[5].Thefewstudiesabouttheeffectofarecolineonc-Myc
inductionhavebeencontroversial.
MicroRNAs(miRNAs)aresmallinterferingRNAsthatactinpost-transcriptionalrepres-
sion.ManystudieshaveindicatedthatarecolinedysregulatesseveralmiRNAs.Recentstudies
havesuggestedthatarecolinecanrepressp53,whichisnecessarytoinducemiR-22expression
[6,7].Inaddition,c-MycalsodirectlysuppressesmiR-22expression[8].Furthermore,miR-22
actsasatumorsuppresserinavarietyofcancers[9,10].However,theroleofmiR-22on
OSCCremainsunknown.
OncostatinM(OSM)isanIL-6familyinflammatorycytokinewhichhasanumber
ofproperties.Itismainlyproducedinneutrophils,Tlymphocytes,macrophagesaswell
ascancercells[11].However,theroleofOSMincarcinogenesisisstilldebated.Some
reportsindicatedthatOSMinhibitstumorgrowthandmetastasisinmelanoma[12],
lungcancer[13],etc.Inversely,OSMhasbeenreportedtoinducetumorgrowthandmetas-
tasisinovariancancer[14],breastcancer[15]andosteosarcoma[16].Thefunctionof
dysregulatedendogenousOSMincancercelllines,includinginOSCCcelllines,isstill
unknown.
Inpresentstudy,wehypothesizedthatarecolineinducesoralcarcinogenesisbyincreasing
c-Mycexpression,consequentlyreducingmiR-22levelscausingdysregulationofOSM.
Thereby,theeffectsofarecolineoncellviabilityandcell-cycleprogressionofOSCCcells
wereinvestigated.ThecorrespondingexpressionsofvarioustargetgenesincludingIL-6,
STAT3,c-MycandmiR-22aswellasOSMwerealsodetermined.Inaddition,theeffectsof
miR-22onpost-transcriptionalrepressionofOSMaswellasmiR-22functionswerestudied
tomoreelucidatemechanismbywhicharecolinemightinfluenceOSCCdevelopment/
progression.
Materialsandmethods
Celllineandcellculture
HumanOSCCcelllines;ORL-48(T)whichiswelldifferentiatedSCCcelllinethatoriginated
frommouth/gumwithnon-betelquidhabitandORL-136(T)whichiswelldifferentiated
SCCcelllinethatoriginatedfromtonguewithbetelquidhabit,kindlyprovidedbyProf.Sok
ChingCheong(CancerResearchInitiativesFoundation,SimeDarbyMedicalCentreJaya,
Malaysia),wereculturedinDMEM/F12(Gibco-LifeTechnologies,GrandIsland,NY,USA)
supplementedwith10%fetalbovineserum(FBS)(Gibco-LifeTechnologies),hydrocortisone
(Sigma-Aldrich,Taufkirchen,Germany)andantibiotics(Gibco-LifeTechnologies)[17].
Humanembryonickidney293FTcellline(HEK293FT,Invitrogen,Carlsbad,CA,USA)was
maintainedinDMEMsupplementedwith10%FBSandantibiotics.Allofthemweremain-
tainedinanincubatorwithanatmosphereat5%CO andat37˚C.
2
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Dysregulatingc-MycandmiR-22,directlytargetingoncostatinMbyarecolineinOSCCcells
Table1. Primersequences.
Primer Oligonucleotidesequence Size(bp)
IL-6 F:50-CTTCGGTCCAGTTGCCTTCT-30 86[18]
R:50-TGGAATCTTCTCCTGGGGGT-30
STAT3 F:50-CTGGCCTTTGGTGTTGAAAT-30 202[19]
R:50-AAGGCACCCACAGAAACAAC-30
c-Myc F:50-CCACTCGGAAGGACTATCCTGCTG-30 152
R:50-GCGCTCCAAGACGTTGTGTGTTCG-30
c-Mycpromoter F:5ʹ-GGTACCTCCTCTCTCGCTAATCTCCGC-3ʹ 468
R:50-AAGCTTCGGGAGGGCTGGGCCAGA-3ʹ
OSM F:50-CTCGAAAGAGTACCGCGTG-30 119[20]
R:50-TCAGTTTAGGAACATCCAGGC-30
miR-22 F:5ʹ-AGCG GACGCAGTGATTTGCT-3ʹ 347
R:5ʹ-AACGTATCATCCACCCTGCT-3ʹ
miR-22cloning F:50-GGTACCAGCGGACGCAGTGATTTGCT-30 359
R:50-GGATCCAACGTATCATCCACCCTGCT-30
OSM3’UTRWT F:5'-CAGTCTAGACATTGATTCAGGGGTCTGATGACAC-3' 433
R:5'- CGTCTAGAAGGGAATCCAAGCAACCGACAGG-3'
OSM3’UTRMut F:5'-GACCTAACTTTACGGAGGTGTAACAGCG-30 428
R:5’-CGCTGTTACACCTCCGTAAAGTTAGGTC-30
Actin F:50-TCACCCACACTGTGCCCATCTACGA-3ʹ 294[21]
R:5ʹ-CAGCGGAACCGCTCATTGCCAATGG-3ʹ
GAPDH F:50-TCATCAGCAATGCCTCCTGCA-3ʹ 117[22]
R:50-TGGGTGGCAGTGATGGCA-3ʹ
https://doi.org/10.1371/journal.pone.0192009.t001
pGL3-Basicvectorcarryingthec-mycpromoter
PCRwasusedtoamplifythec-myccorepromoterfromHeLagenomicDNAusingthec-Myc
promoterprimerasshowninTable1.PCRconditionsaredescribedinSupportinginforma-
tion:S1Table.The468bpPCRproductwaspurifiedusingaHiYield™Gel/PCRDNAFrag-
mentsExtractionKit(RBCBioscience,Taipei,Taiwan)andclonedintopGEM-Tvector
(Promega,Madison,WI,USA).TheconstructedplasmidwastransformedintoEscherichiacoli
(E.coli)strainDH5α.Theproductcontainingc-myccorepromoterinpGEM-Tvectorwas
subclonedintothepGL3-Basicvector,whichlackseukaryoticpromotersequencesandcon-
tainsthefireflyluciferase(Promega)asareporter.Thec-myccorepromotersequencewas
confirmedbysequencinganalysis.
pIRES-miR-22vector
ThemiR-22fragmentcontainingthestem-loopsequencewasamplifiedfromORL-48(T)
cDNAusingspecificprimersasshowninTable1:Hsa-miR-22-forwardandreverseprimers
(withKpnIandBamHIrestrictionsite,respectively).PCRconditionsaredescribedinSupport-
inginformation:S1Table.ThefragmentwasclonedintopGEM-Tvectorandthensubcloned
intopIRES2-EGFPvector(Clontech,PaloAlto,CA,USA).
pGL3-OSM3ʹUTR(untranslatedregion)WTandMutvectors
OSM3ʹUTRWTwereamplifiedfromORL-48(T)cDNAusingspecificprimerswithXbaI
restrictionsite(Table1).ThemutantofOSM3ʹUTRwasamplifiedbyPCR-basedsite-directed
mutationusingOSM30-UTRMut-forwardandreverseprimers.PCRconditionsaredescribed
inSupportinginformation:S1Table.BothOSM3ʹUTRWTandMutwereclonedinto
pGEM-TvectorandthensubclonedintoXbaI-digestedpGL3-Controlvector(Promega).
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Dysregulatingc-MycandmiR-22,directlytargetingoncostatinMbyarecolineinOSCCcells
MTTassayfordeterminationofcellcytotoxicityandcellproliferation
Todeterminecytotoxicityofarecoline,4x104cellsofORL-48(T)orORL-136(T)cellswere
seededintoeachwellof96-welltissuecultureplates,andmaintainedincompletemediumfor
24hours.Thecellsweretreatedwitharecoline(Sigma-Aldrich,St.Louis,MO,USA)at0,25,
50,100,200,400,800and1,200μg/mlintriplicatefor24hours.Cellviabilitywasdetermined
byMTTassay.
Todeterminetheeffectofarecolineoncellproliferation,5x103cellsofORL-48(T)and
ORL-136(T)cellsinserum-starvedDMEM/F12mediumwereseededintoeachwellof96-well
tissuecultureplatesfor24hours.Thecellsweretreatedwitharecolineat0,0.025,0.25,2.5and
25μg/mlinserum-starvedDMEM/F12mediumintriplicateforafurther24hours.Cellprolif-
erationwasdeterminedbytheMTTassay.
ByMTTassay,10μlMTT(5mg/ml)wasaddedtoeachwell.After4hours,themedium
wasremovedandthewater-insolublepurpleformazanparticlesweredissolvedin100μl
DMSOsolution.Theabsorbancewasreadat570nmwithaMicroplateReader(TECAN,Salz-
burg,Austria).
Flowcytometryforcell-cycleanalysisinarecoline-treatedandmiR-
22-transfectedORL-48(T)cells
Inarecoline-treatedcells,105ORL-48(T)cellswereseededinto6-welltissuecultureplates.
Cellsweresynchronizedbyserumstarvationfor24hoursthentreatedwith0,0.025and25μg/
mlarecolinefor24hours.FormiR-22-treatedanduntreatedcells,105ORL-48(T)cellswere
transfectedwithmockcontrolandpIRES-miR-22for6hoursandthentransfectedcellswere
culturedincompletemedium(DMEM+10%FBS)andincubatedfor24hours.Inbothexper-
iments,cellsweredetachedwith0.25%trypsinsolution,washedtwicewithphosphate-buffered
saline(PBS)andfixedwith70%ethanolat4˚Cfor24hours.Propidiumiodide(PI)solution
containing1Xbindingbuffer,20μg/mlPI(BDPharmingen,Heidelberg,Germany)and0.5U
RNaseA(Sigma)wasfreshlypreparedtostainthecells.Afterincubationfor20minutes,cells
wereanalyzedusingflowcytometry(BectonDickinsonFACSCantoII,USA).Triplicateinde-
pendentexperimentswereperformed.
RT-PCRfordeterminationofIL-6,STAT3,c-Myc,miR-22andOSM
expression
RNAwasextractedfromcellsincludingarecoline-treated/untreatedcells;ORL-48(T)and
ORL-136(T)cellsandmiR-22-transfected/untransfectedcells;ORL-48(T)andORL-136(T)
cellsusingTRIzol1reagent(Invitrogen,Carlsbad,CA).cDNAwassynthesizedfromthe
extractedRNAwithanOligo(dT)primerusingaSuperScriptIIIFirst-StrandSynthesisSys-
tem(Invitrogen)accordingtothemanufacturer’sinstruction.ThecDNAwasusedasthe
templatetodeterminetheexpressionofOSM,IL-6,STAT3,c-MycandmiR-22using
RT-PCR.GAPDH(forOSM,IL-6,STAT3andc-Myc)andβ-actin(formiR-22)wereuseas
internalcontrols.Eachreactionconsistedof4μlofcDNA,1XSsoAdvanced™Universal
SYBR1GreenSupermix(Bio-RadLaboratories,Hercules,CA),300nMsenseand300nM
antisenseprimers.TheRT-PCRwasperformedintheLightCycler480(RocheAppliedSci-
ence,Indianapolis,IN,USA).FoldchangeexpressionwascalculatedusingΔΔCtandrela-
tivetotheuntreatedgroup.TheprimersequencesandRT-PCRconditionswereshown
inTable1andSupportinginformation:S2Table.Eachexperimentwasperformedin
triplicate.
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Dysregulatingc-MycandmiR-22,directlytargetingoncostatinMbyarecolineinOSCCcells
Westernblottingfordeterminationofc-MycandOSMprotein
Proteinfromarecoline-treatedanduntreated,andmiR-22transfectedORL-48(T)andORL-
136(T)cellswasisolatedwithradioimmunoprecipitationassay(RIPA)buffer(25mMTris-
HClpH7.6,150mMNaCl,1%sodiumdeoxycholate,0.1%SDS).OSM,c-Mycandactinpro-
teinsweredeterminedbywesternblotusingasprimaryantibodiesrabbitanti-OSM(1:100
dilution;Sigma),mouseanti-c-Myc(1:100dilution;clone9E10,SantaCruzBiotechnology,
SantaCruz,CA,USA)andmouseanti-actin(1:1,000dilution;Sigma).Theintensityofprotein
bandswasmeasuredusingImageJ1.49vsoftware(NationalInstitutesofHealth,Bethesda,
MD,USA).
Determinationofarecolinetargetingc-mycpromoteractivity
500ngpGL3-c-mycpromoter(cMYCP)orpGL3-Basic(mock)vectorwastransfectedinto4x
104ORL-48(T)cellsin96-wellplatesfor6hoursandthenmaintainedinDMEMcomplete
mediumwith/withoutarecolinetreatmentfor24and48hours.c-mycpromoteractivitywas
measuredbyluciferaseassayusingBright-Glo™system(Promega).Theluciferaseactivityunit
inpGL3-cMYCP-transfectedcellswasnormalizedwitharecoline-treatedmockcontrolsand
relativetotheuntreatedgroup.Thisexperimentwasperformedintriplicate.
LuciferaseassayfordeterminationofmiR-22targetingOSM30UTR-WT
andOSM30UTR-Mut
250ngpIRES-miR-22and100ngpGL3-OSM30UTRWTorMutvectorswereco-transfected
into293FTcells.At24and48hoursposttransfection,luciferaseactivityinco-transfected
cellswasmeasuredusingtheBright-Glo™system.LuciferaseactivityofpIRES-miR-22and
pGL3-OSM30UTRWTorMutco-transfectedcellswasnormalizedagainstluciferaseactivity
incellsco-transfectedwithpIRES2-miR-22-andpGL3-Controlvectors,andwasrelativeto
thenormalizedluciferaseactivityofpIRES2-EGFPandOSM30UTRco-transfectedcellswith
pIRES2-EGFPandpGL3-controlco-transfectedcells.Thisexperimentwasperformedin
triplicate.
Woundhealingassayfordeterminationofcellmigration
2x105ORL-48(T)cellswereseededinto24-welltissuecultureplatesfor24hoursandtrans-
fectedwithpIRES-miR-22vectorusingLipofectamine12000.Aftertransfectionfor6hours,
themonolayerwasgentlyandslowlyscratchedwitha10μlpipettetip.Woundclosurewas
determinedat0,24,48and72hoursunderamicroscope.Extentofwoundclosurewasmea-
suredusingNIS-ElementsAdvancedResearchImagingSoftwareversion3.0.Thecellmigra-
tionassaywasperformedintriplicateinseparatewells.
Statisticalanalysis
Dataareexpressedasmean±SEM(standarderrorofthemean).(cid:3),(cid:3)(cid:3)and(cid:3)(cid:3)(cid:3)weredenotedas
significantdifferenceinP<0.05,0.01and0.001,respectively.Pairedt-testwasusedforcell-
cycleanalysis.One-wayANOVAfollowedbyTukey’smultiplecomparisontestwasusedto
analyzecellviabilityandRT-PCRresultsinarecoline-treatedand-untreatedcells.Two-way
ANOVAwasusedtoanalyzethesignificantlevelofluciferaseactivitybetweenandwithin
groups.AllstatisticalanalysiswasperformedusingPrism5software(GraphPad,SanDiego,
CA,USA).
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Dysregulatingc-MycandmiR-22,directlytargetingoncostatinMbyarecolineinOSCCcells
Results
Highdosesofarecolinewerecytotoxicbutlowdosesinducedcell
proliferationandcell-cycleprogression
Toinvestigatethecytotoxicityofarecoline,ORL-48(T)andORL-136(T)celllinesweretreated
withdifferentconcentrationsofarecolinefor24hours.Fig1Aand1Bshowedcytotoxiclevels
ofarecolineonORL-48(T)andORL-136(T)cellsthatwerehigherthan100and200μg/ml,
respectively.ArecolineatlowconcentrationincreasedviabilityofbothORL-48(T)andORL-
136(T)cells(Fig1Cand1D).Theseresultsdemonstratedthatarecolineat0.025μg/ml
increasedcellviabilityofOSCCcelllines,therefore,thisconcentrationwasusedinfurther
experiments.
Theeffectofarecolineoncell-cycleprogressionwasconfirmedbyflowcytometry.Areco-
lineat0.025μg/mlinducedsignificantproliferationofORL-48(T)cellsbyincreasingthepro-
portionofG2/Mphase(8.9±2.8%G2/Mcells)whencomparedtountreatedcells(3.9±1.2%
G2/Mcells)asshowninFig1E,1Fand1G.
Theeffectsofarecolineonc-mycpromoterandexpression
c-Mycisatranscriptionalactivatorandrepressorofvarioustargetgenes,contributingto
manybiologicalprocessesespeciallycellproliferation[5].c-Mycisalikelytargetforarecoline.
However,theeffectofarecolineonc-Mycexpressionisdebated.Inordertoexplorethiseffect,
expressionofc-Mycinarecoline-treatedORL-48(T)cellswasassayedusingrealtimepolymer-
asechainreaction(RT-PCR)andwesternblot.Inthearecoline-treatedcells,c-Mycexpression
wasincreasedatboththemRNAandproteinlevels(Fig2Aand2B).Thelevelofc-Myc
mRNAandproteinincellstreatedwith0.025μg/mlarecolinewassignificantlyhigherthanin
theothertreatments(Fig2Cand2D).Thisindicatesthatarecolinecanupregulatec-Myc
expression.
Toconfirmthis,apGL3-Basicvectorcontainingthec-myccorepromoter(P1andP2
regions)wasconstructed.ORL-48(T)cellsweretransfectedwiththisvector.From24hours
post-transfection,thecellswereincubatedwithvariousconcentrationsofarecolinefor24and
48hours.Arecolinewasshowntoinducetranscriptionalactivityofthec-mycpromoteras
showninFig2E.At0.025μg/mlarecolinetreatment,relativeluciferaseactivitywassignificant
higherthaninuntreatedcellsfor24and48hours.Thisdemonstratesthatthelowconcentra-
tion(0.025μg/ml)ofarecolinecouldinducetranscriptionalactivityofc-mycpromoter,result-
inginc-Mycupregulation.However,athigherconcentrationsofarecoline,transcriptional
activityofthec-mycpromoterwasdecreased.
ArecolinecaninduceIL-6/STAT3upstreamofc-Myc
IL-6/STAT3signalingcascadeinducesmanydownstreamtargetsanditsdysregulationcould
contributetoinitiation,promotion,andprogressionoftumor-associatedinflammation[23].
c-Mycisawell-knowntargetofIL-6/STAT3[5].Previousstudiesrevealedthatarecanut
extractcouldinduceIL-6production,whilearecolinedecreasedIL-6levels[24].Inourfind-
ing,alowconcentrationofarecolinecouldinducec-Myctranscriptionalactivity.Tomore
clarifythepossibleinvolvementofIL-6andSTAT3,therefore,ORL-48(T)andORL-136(T)
cellsweretreatedwitharecolineandinvestigatedforIL-6andSTAT3expressionusing
RT-PCR.ExpressionofbothIL-6andSTAT3inORL-48(T)seemedtodecreaseincellstreated
with25μg/mlofarecoline,whereasitwassignificantlyhighestincellstreatedwith0.025μg/
mlarecolineinbothORL-48(T)andORL-136(T)cells(Fig3).Thisresultindicatedthatdiffer-
entarecolineconcentrationsandcelltypesmayimpactexpressionofitstargetgenes.At
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Dysregulatingc-MycandmiR-22,directlytargetingoncostatinMbyarecolineinOSCCcells
Fig1.Theeffectsofarecolineoncellviabilityandcell-cycleprogression.Cytotoxicity(AandB)andcell
proliferation(CandD)weredeterminedinarecoline-untreatedortreatedOSCCcelllinesatvariousconcentrations
for24hoursusingtheMTTassay.Statisticalsignificanceofthedifferencesofcellviability(%)wasanalyzedusingOne-
wayANOVAfollowedbyTukey’smultiplecomparisontest((cid:3)P<0.05,(cid:3)(cid:3)P<0.01and(cid:3)(cid:3)(cid:3)P<0.001).Cell-cyclephase
distribution(EandF)inORL-48(T)cellstreatedwith0and0.025μg/mlofarecolineinsynchronizedconditionwas
analyzedbyflowcytometry.ThepercentagesofG0/G1,SandG2/Mpopulation(G)ofarecoline-treatedcellswere
comparedtountreatedORL-48(T)cellsascontrol.StatisticalsignificanceofthedifferencesofG2/Mpopulationwas
analyzedusingPairedt-test((cid:3)P<0.05).
https://doi.org/10.1371/journal.pone.0192009.g001
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Dysregulatingc-MycandmiR-22,directlytargetingoncostatinMbyarecolineinOSCCcells
Fig2.Theeffectsofarecolineonc-Mycexpressionandtranscriptionalactivity.ORL-48(T)cellstreatedwith0,0.025and25μg/mlofarecolinefor24
hourswereassayedtodeterminelevelsofc-MycexpressioninmRNA(RT-PCR)(A)andprotein(westernblot)(B).Relativec-Mycexpressionand
relativeintensityofc-MycproteinbandwereinvestigatedinRT-PCRandwesternblotresult(C-D).Statisticalsignificanceofthedifferenceswas
analyzedusingOne-wayANOVAfollowedbyTukey’smultiplecomparisontest((cid:3)P<0.05,(cid:3)(cid:3)P<0.01and(cid:3)(cid:3)(cid:3)P<0.001).MockorpGL3-cMYCP
vector-untransfectedortransfectedORL-48(T)cellsweretreatedwith0,0.025,0.25and25μg/mlofarecolinefor24and48hours(E).The
transcriptionalactivityofthec-mycpromoterwasdeterminedbyluciferaseactivity.Statisticalsignificanceofthedifferencesofluciferaseactivitywas
analyzedusingTwo-wayANOVA((cid:3)P<0.05,(cid:3)(cid:3)P<0.01and(cid:3)(cid:3)(cid:3)P<0.001).
https://doi.org/10.1371/journal.pone.0192009.g002
0.025μg/mlarecolinecouldinduceIL-6/STAT3expression,possiblycausingupregulationof
thedownstreamtarget,c-Myc.
TheeffectofarecolineonmiR-22expressioninOSCCcelllines. Somestudieshave
demonstratedthatarecolinehadacomprehensiveeffectoncellulargeneexpression,including
expressionofmiRNA[25].TheroleofarecolineinmiRNAexpressionhasreceivedlittleinves-
tigation.Apreviousstudyhavesuggestedthatarecolinerepressedexpressionofp53[26],a
proteinthatdirectlyupregulatesmiR-22[10].Inversely,c-MycdirectlyinhibitedmiR-22
expression[8].Inaddition,theroleofmiR-22inOSCChasremainedunclear.Therefore,to
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Dysregulatingc-MycandmiR-22,directlytargetingoncostatinMbyarecolineinOSCCcells
Fig3.EffectofarecolineonIL-6andSTAT3inORL-48(T)andORL-136(T)cells.ORL-48(T)andORL-136(T)
cellsweretreatedwith0,0.025and25μg/mlarecolinefor24hours.ExpressionofIL-6(AandD)andSTAT3(Band
E)wereinvestigatedbyRT-PCRandtheirampliconswerevisualizedby2%agarosegelelectrophoresis(CandF).
StatisticalsignificanceofthedifferencesofrelativeexpressionwasanalyzedusingOne-wayANOVAfollowedby
Tukey’smultiplecomparisontest((cid:3)P<0.05and(cid:3)(cid:3)P<0.01).
https://doi.org/10.1371/journal.pone.0192009.g003
moreelucidatetheroleofarecolineinepigeneticalterationespeciallytumorsuppressingmiR-
22inOSCC,ORL-48(T)andORL-136(T)cellsweretreatedwitharecolineat0,0.025and
25μg/mlandthelevelofpri-miR-22wasexaminedbyRT-PCR.Incellstreatedwith0.025and
25μg/mlofarecoline,miR-22expressionwassignificantlyreduced(Fig4Aand4B),incon-
trasttountreatedcells.ThisresultindicatedthatmiR-22couldbesuppressedbyarecolinein
OSCC.
PLOSONE|https://doi.org/10.1371/journal.pone.0192009 January31,2018 9/16
Dysregulatingc-MycandmiR-22,directlytargetingoncostatinMbyarecolineinOSCCcells
Fig4.TheeffectsofarecolineonmiR-22andOSMexpression.BothORL-48(T)andORL-136(T)cellswereexposedto
0,0.025and25μg/mlofarecolinefor24hours.Pri-miR-22expressionwasdetectedbyRT-PCR.β-actinwasusedas
internalcontrols(A-B).ORL-48(T)cellsweretreatedwithvariousconcentrationsofarecolinefor24hours;thenOSM
mRNAexpressioninthesecellswasanalyzedusingRT-PCR(C-D).OSMproteinlevelsinORL-48(T)(E)andORL-136
(T)(F)cellswereexaminedbywesternblotandrelativeintensitieswereanalyzedbyImageJ1.49vsoftware(G-H).
StatisticalsignificanceofthedifferencesofrelativeexpressionwasanalyzedusingOne-wayANOVAfollowedbyTukey’s
multiplecomparisontest((cid:3)P<0.05,(cid:3)(cid:3)P<0.01and(cid:3)(cid:3)(cid:3)P<0.001).
https://doi.org/10.1371/journal.pone.0192009.g004
OSMwasaputativetargetofmiR-22andupregulatedbyarecoline
Frominsilicoresults,OSMispredictedasatargetofmiR-22accordingtoalgorithmsinTar-
getScanHumanRelease6.2[27]andmiRNA.org[28].Interestingly,OSMpromotedtumor
growthandprogressioninseveralcancers[11].OSMinducedIL-6andSTAT3,withsubse-
quenteffectsonmanysignalingcascades[29]andalsoinducedc-Mycexpression[30].More-
over,dysregulationofc-MycswitchedOSMfunctionfromcancersuppressiontocancer
promotionbecauseOSM-inducedsenescencewasinhibitedbyc-Myc[11].Therefore,we
PLOSONE|https://doi.org/10.1371/journal.pone.0192009 January31,2018 10/16