MicroRNA-1298 is regulated by DNA methylation and affects vascular smooth muscle cell function

12 Pages·2015·1.64 MB·English

CardiovascularResearch(2015)107,534–545
doi:10.1093/cvr/cvv160
MicroRNA-1298 is regulated by DNA methylation
and affects vascular smooth muscle cell function
by targeting connexin 43
D
Wei Hu1†, Mian Wang1†, Henghui Yin1, Chen Yao1, Qiong He2, Leping Yin3, ow
n
Chunxiang Zhang4, Wen Li5, Guangqi Chang1*, and Shenming Wang1* loa
d
e
d
1DivisionofVascularSurgery,TheGuangdongEngineeringLaboratoryforDiagnosisandTreatmentofVascularDiseases,TheVascularSurgicalDiseaseResearchCenterofGuangdong fro
Province,FirstAffiliatedHospital,SunYat-senUniversity,58ZhongShanErRoad,Guangzhou,Guangdong510080,China;2DepartmentofPathology,FirstAffiliatedHospital,SunYat-sen m
University,Guangzhou,China;3DivisionofVascularSurgery,SichuanAcademyofMedicalSciencesandSichuanProvincialPeople’sHospital,Chengdu,China;4DepartmentofPharmacology, h
RushUniversityMedicalCenter,Chicago,USA;and5LaboratoryofGeneralSurgery,FirstAffiliatedHospital,SunYat-senUniversity,Guangzhou,China ttps
Received26August2014;revised29April2015;accepted7May2015;onlinepublish-ahead-of-print29May2015 ://ac
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Timeforprimaryreview:20days m
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Aims Growing evidencelinksmicroRNAtotheprocessof peripheral vasculardisease.Recently,we have found that o
m
microRNA-1298(miR-1298)isoneofthemostsignificantlydown-regulatedmicroRNAsinhumanarterieswithar- /c
a
teriosclerosisobliterans(ASO)ofthelowerextremities.However,littleisknownregardingitsroleintheprocess rd
io
ofASO.Thepresentstudyaimedtoinvestigatetheexpression,regulatorymechanisms,andfunctionsofmiR-1298 v
a
s
intheprocessofASO. c
..................................................................................................................................................................................... re
s
Methods Usingquantitativereverse-transcriptionPCRandinsituhybridizationassays,miR-1298wasobservedpredominantly /a
andresults expressedinthevascularsmoothmusclecells(VSMCs)andwassignificantlydown-regulatedinASOcompared rticle
withnormalarteries.PyrosequencinganalysisrevealedthatthemiR-1298DNAupstreamofCpGsiteswerehyper- -a
b
s
methylatedinASOcomparedwithnormalarteries.Next,theluciferasereporterassayrevealedthatmiR-1298 tra
c
down-regulationisrelatedwithupstreamDNACpGsitehypermethylation.IntroducingamiR-1298mimicintocul- t/1
turedVSMCssignificantlyattenuatedcellproliferationandmigration.Connexin43(Cx43)wasvalidatedtobeafunc- 07
/4
tionaltargetofmiR-1298thatwasinvolvedinthemiR-1298-mediatedcellulareffects.Finally,lentivirus-mediated /5
3
deliveryofmiR-1298anditstargetCx43intoaratcarotidballooninjurymodelindicatedthatre-overexpressionof 4
/3
miR-1298significantlydecreasedneointimalformationbytargetingconnexin43. 1
1
..................................................................................................................................................................................... 4
7
Conclusion OurdatademonstrateaspecificroleoftheupstreamDNAmethylation/miR-1298/Cx43pathwayinregulatingVSMC 8
b
--------------------------f-u-n--c-t-io--n--a-n--d--s-u-g-g--e-s-t--th--a-t--m--o-d--u-l-a-t-io--n--o-f--m--i-R---1--2-9-8--l-e-v-e--ls--m--a-y--o--f-fe--r-a--n--o-v-e--l-t-h-e--r-a-p-e--u-t-ic--a--p-p-r-o--a-c-h--f-o--r-A--S--O--.------------ y gu
e
Keywords Arteriosclerosisobliterans † Smoothmusclecell † microRNA † Connexin43 † Methylation st o
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1. Introduction wallintotheintimalspaceandthenprogressivelyformaneointima. 0
1
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Thisformationtogetherwiththepresenceofabundantlevelsofextra-
Arteriosclerosis obliterans (ASO) is one of the most significant cellularmatrixproteinsleadstovascularstenosis.6However,themo-
health-relatedissuesandremainstheleadingcauseoflimblossworld- lecularmechanismsunderlyingVSMCproliferationandmigrationin
wide.1,2SinceendovascularsurgerywasfirstusedtotreatASO,vascu- ASOremainunclear.
lar restenosis has been the main limiting factor for the long-term MicroRNAs are endogenous, single-stranded, small noncoding
successofrevascularizationprocedures.3Inrecentdecades,manyre- RNAs.Theyaregenerallyregardedasnegativeregulatorsofgeneex-
portshaveshownthatexcessivevascularsmoothmusclecell(VSMC) pressionbecausetheystimulatethedegradationorsuppressthetrans-
proliferationandmigrationplayimportantrolesinthepathogenesisof lationoftargetmRNAsbycomplementarybasepairingtosequences
ASO.4,5Normally,quiescentVSMCsmigratefromthemedialarterial within the 3′-UTR region of protein-coding mRNA transcripts.7
*Correspondingauthor.Tel/Fax:+8620877557666212,Email:[email protected](G.C.);[email protected](S.W.).
†Theseauthorscontributedequallytothiswork.
PublishedonbehalfoftheEuropeanSocietyofCardiology.Allrightsreserved.&TheAuthor2015.Forpermissionspleaseemail:[email protected].
miR-1298AffectsVSMCFunctionbyTargetingCx43 535
Currently,morethan2000humanmicroRNAshavebeenclonedand miR-1298upstreamDNACpGsites.Allrelatednucleicacidsequences
sequenced,andthesemicroRNAsareimportantregulatorsofabroad wereverifiedintheGenBankdatabase.
rangeofbiologicalprocesses,includingmetabolism,cellproliferation,
2.8 Luciferase reporter assay
embryonicdevelopment,homeostasis,apoptosis,andpossiblythedis-
easemechanismsofarteriosclerosis.8 After close examination of miR-1298 precursor gene and analysis of
ThemicroRNAexpressionprofileinratvesselshasbeendescribed miR-1298upstreamDNAsequencepromoteractivityusingbioinformatics
byJietal.,9andthemicroRNAexpressionprofileofhumanarteries methods(JASPARdatabase),11weidentifiedmiR-1298-associatedCpG
withASOwasoriginallydescribedinanearlierstudybyourgroup.10 siteslocatedat21900to+100upstreamofthemiR-1298precursorre-
gion.The2kbDNAfragmentwasclonedintoapGL3vector.Renillaluci-
We found that certain microRNAs such as miR-21, miR-575,
ferase vector pRL-SV40 was co-transfected in each experiment as an
miR-1826,andmiR-22areup-regulatedinarterieswithASO.Incon-
internalcontrol.A3′-UTRluciferaseassaywasperformedtovalidate
trast,somemicroRNAssuchasmiR-1298,miR-100,miR-125b,and
Cx43asatargetgeneofmiR-1298.
miR-24aredown-regulatedinthesehumanASOarteries.Wefound D
o
thatmiR-1298wasoneofthemostsignificantlydown-regulatedmicro- 2.9 Fluorescencerecoveryafterphotobleaching wn
RNAsinASO.Ourcurrentstudyfocussedonthepotentialrolesof lo
(FRAP) technique a
d
miR-1298inVSMCfunctioninvivoandinvitroandthemechanisms e
involved. F(GRJAICP)wbeatswueseendVtSoMmCesa.sure gap junction intercellular communication d fro
m
h
2. Methods 2.10 Lentiviral transduction ttp
s
A lentiviral transduction system was used to overexpress Cx43 or ://a
PleaseseeSupplementarymaterialonlinefordetailedmethods. ca
miR-1298. d
e
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2.1 Sample acquisition
ic
2.11 Rat carotid artery balloon injury model .o
ThisstudywasapprovedbytheResearchEthicsCommitteeoftheFirstAf- u
p
filiatedHospitalofSunYat-senUniversityandconformedtotheDeclar- AnSDratcarotidarteryballooninjurymodelwasusedtotesttheabilityof .c
o
ationofHelsinki.ASOarterysampleswereobtainedfrompatientswith miR-1298toinhibitthedevelopmentofvascularneointima.Thedetailed m
ASOwhounderwentlowerlimbamputation,andnormallowerlimbartery methodfortheratcarotidarteryballooninjurymodelisdescribedinSup- /ca
sampleswereacquiredfromdonorswithoutASO.Consentwasobtained plementarymaterialonline.9 rdio
v
fromeitherthedonorsortheirfamilymembers. a
s
2.12 Statistics c
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2.2 Cell culture s
StatisticalanalysiswasconductedwithStudent’sttestforaverage(mean) /a
VSMCsweremaintainedinDMEMwith10%fetalbovineserumand1% comparisonsorone-wayANOVAformultiplecomparisonsfollowedby rtic
le
penicillin/streptomycinat378Cinahumidifiedincubatorwithanatmos- theS–N–Ktesttoidentifysignificantdifferences.Therelationshipbe- -a
pherecontaining5%CO2. tPw-veaelunetsh,ev0a.r0i5abwleesrweacsodnesitdeerrmedinesidgnbiyficthanetP.eAalrlsdoantacworerreelaptirooncecsoseefdficuiseinntg. bstra
c
2.3 Quantitative reverse-transcription GraphPadPrism5.0andSPSSsoftware,version17.0. t/1
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PCR assay 7
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QuantitativeReverse-transcriptionPCR(qRT-PCR)assaywasperformed 3. Results /53
4
todetectmiR-1298andCx43mRNAlevelsasdescribedinSupplementary /3
1
materialonline. 1
3.1 The expression of miR-1298 was 4
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2.4 In situ hybridization and decreased in ASO arteries b
y
immunohistochemistry/IF g
OurpreviousstudyrevealedthatmiR-1298isoneofthemostsignifi- u
e
In situ hybridization (ISH) was used to determine the localization of cantlydown-regulatedmicroRNAsinASOarteriescomparedwith st o
miR-1298.Immunohistochemistry(IHC)/IF(IFC)wasperformedtodetect normalarteries.Inthepresentstudy,usingaqRT-PCRassay,wecon- n 0
smoothmusclea-actin(SM-a-actin),connexin43(Cx43),andKi67. firmedthatmiR-1298wassignificantlydown-regulatedinASOarteries 4 A
2.5 Western blot assay dcoismtrpibaurteidonwoitfhmnoiRrm-1a2l9a8rtienritehsebayr(cid:3)te6ry4%w(aFlli,guwree1diAs)s.eTcoteddeAteSrOminarettehrey pril 20
Western blot assay was performed to detect Cx43, Ki67, GAPDH, wallsintothreelayersanddetectedmiR-1298expressionusingthe 19
SM-a-actin,tublin,calponinproteinlevelsasdescribedinSupplementary
qRT-PCRassay.AsshowninFigure1B,miR-1298wasexpressedpri-
materialonline.
marilyinarterymediaratherthantheintimaandadventitia.Tofurther
2.6 Analysis of VSMC proliferation, migration, investigatethecellulardistributionofmiR-1298inthearterywall,we
cell cycle, and apoptosis performedco-IFwithsmoothmusclemarkerSM-a-actin.Asexpected,
miR-1298(redcolour)wasfoundco-localizedwithSM-a-actin(green
Cellcountingkit-8andEdUassayswereusedtodetectcellproliferation,
colour) in the artery media (Figure 1D). This finding indicatesthat
whiletranswellandwoundclosureassayswereperformedtodetectcell
miR-1298ismainlyexpressedinVSMCs.Next,theintegraloptical
migration.Thecellcycleandapoptosisanalyseswereperformedusing
flowcytometry. density(IOD)value(Figure1C)ofmiR-1298staininginbothnormal
andASOarterysectionswascalculated.TheIODvalueofmiR-1298
2.7 Pyrosequencing analysis wasfoundtobesignificantlydecreased(by(cid:3)52%)intheASOartery
Pyrosequencing,oneofthemostaccuratemethodsavailableforquantifying sectionscomparedwiththenormalarterysections.Thisresultsuggests
DNAmethylation,wasutilizedtoevaluatethemethylationstatusofthe thatmiR-1298isdown-regulatedinASOVSMCs.
536 W.Huetal.
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Figure1 ThecharacteristicsofmiR-1298expressionandtheeffectofDNAmethylationonit.(A)miR-1298expressioninbothASOandnormal
arterieswasdeterminedbyqRT-PCR(n¼8forboth).(B)miR-1298expressioninthethreelayersoftheASOarterywallwasdeterminedbyqRT-PCR
(n¼6).(C)IODcalculationsforthemiR-1298-stainedarterysectionsrevealedthatmiR-1298stainingwassignificantlydecreasedintheASOsections
(n¼16)comparedwiththenormalsections(n¼8).(D)Co-stainingofmiR-1298(red)andSM-a-actin(green)inhumanarterysections.ISHshowed
thatmiR-1298localizedinarterialVSMCs.(E)Thepri-miR-1298levelwasmarkedlylowerinASOarteriesthannormalarteries(n¼6forboth).(F)A
schematicillustrationofthelocationoftheCpGsitesforpyrosequencing.(G)ThemethylationrateofmiR-1298DNAupstreamofCpGsitessignificantly
differedbetweennormalandASOarteries(n¼8forboth),asdeterminedbypyrosequencinganalysis.(H)TheDNAmethyltransferaseinhibitor
5-aza-2′-deoxycytidine(5-aza,10mmol/L)inhibitedthemethylationofrelatedCpGsites(72h)invitro(n¼3).(I)5-azaup-regulatedtheexpression
ofpri-miR-1298inVSMCs(n¼5).(J)5-azaup-regulatedtheexpressionofmiR-1298inVSMCs(n¼5).(K)TheclonedmiR-1298DNAupstream
of2000bpfragment(pGL3-miR-1298)showedactivity,whereasthepGL3-basiccontroldidnot.WhentheCpGsitesinthefragmentweremutated
bymethylase,therelativeluciferaseactivityofmut-pGL3-miR-1298wasdown-regulatedcomparedwithpGL3-miR-1298.Themethyltransferaseinhibi-
tor5-azaup-regulatedtheactivityofthemutatedfragment(n¼3).Scalebar¼200mm;*P¼0.05,**P¼0.01,***P¼0.001.
miR-1298AffectsVSMCFunctionbyTargetingCx43 537
3.2 miR-1298 deficiency in ASO arteries intheSphase(Figure2L),moderatelyinducedcellproliferationandmi-
was related with DNA upstream of CpG gration(Figure2MandN),butdidnotaffectcellapoptosisinVSMCs
(Figure2O).Inaddition,thesequenceofmiR-1298wasfoundtobecon-
site methylation
servedinmanyspecies(Supplementarymaterialonline,FigureS1)and
First,thelevelofprimary-miR-1298(pri-miR-1298)inbothnormaland
miR-1298wasalsoabletoinhibitratVSMCproliferationandmigration
ASOarterieswasdetectedbyqRT-PCRassay.Thepri-miR-1298level
(Supplementarymaterialonline,FigureS2).Theseresultsconfirmthat
wassignificantlylowerinASOarteriesthannormalarteries(Figure1E),
miR-1298mightinhibitVSMCproliferationandmigration.
suggestingthatmiR-1298isdown-regulatedatthetranscriptionallevel
in ASO arteries. Although DNA methylation-mediated epigenetic 3.4 Cx43 is a direct target of miR-1298
silencingofmicroRNAshaspreviouslybeendemonstrated,thespecific
Cx43(connexin43)wasselectedasapotentialmiR-1298targetbased
mechanismbywhichmiR-1298issilencedinASOisunknown.Toin-
onitsmRNA3′-UTRcomplementarytomiR-1298(Figure3A).Cx43
vestigatethetranscriptionalregulationofmiR-1298expression,weex-
aminedits5′genomicsequenceandidentifiedaCpG-richfragmentin participatesinregulatingVSMCproliferationandmigrationandplays Do
theregioncovering 222to +25bpupstreamofthemiR-1298core animportantroleintheprocessofatherosclerosis.12Interestingly, wnlo
wefoundthattheCx43proteinlevelwassignificantlyhigherinASO a
sequence(Figure1F).Then,themethylationstatusofthesequence d
wshaoswdeetdecthteadtuthsiengdpeygrroeseeqoufemnceitnhgyalastsiaoyn.Twhaesp(cid:3)yr6o3se.1q2u%enicnintghaenaAlySsOis baryteIFrCiesancdomwpesatreerdnwbliotht)n.Oornmtahleacrotenrtireasry(F,itghuerem3iRB-–1D29,8asledveetlewrmasinseigd- ed from
arteriescomparedwith31.29%innormalarteries(Figure1G).Thisdif- n(Fifiigcuarent3lyB,loaswdeerteinrmAinSeOdbaryteFIrSieHs).cTohmepparreesdenwtirthesunlotsrmcoanlfiarrmteerdieas http
ference suggests that methylation may be essential for the down- s
regulation ofmiR-1298inASO.Wenext treatedcultured VSMCs negativecorrelationbetweentheexpressionofCx43andmiR-1298 ://ac
with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine in the in vivo setting (Figure 3E). Moreover, in cultured VSMCs, ad
e
miR-1298significantlyreducedCx43proteinexpression,whilethe m
(d5e-carzeaa)seadnd(Ffoiguunred1thHa)tatnhde dtheegreeexporefsCsipoGn osifteprmi-emthiRy-la1t2io9n8 wanads miR-1298inhibitormoderatelyincreasedCx43proteinexpression ic.ou
(Figure3FandG).Inaddition,miR-1298slightlydown-regulatedthe p
miR-1298wassignificantlyup-regulated(Figure1IandJ).Wethenrea- .c
Cx43mRNAlevel(Figure3H).ThesedatasuggestthatCx43might o
sonedthatmiR-1298down-regulationinASOisassociatedwithDNA m
beadirecttargetofmiR-1298.TodeterminewhethermiR-1298dir- /c
methylation. a
ectlybindsCx43mRNAandregulatesitsexpression,thetwoputative rd
ToelucidatethelinkbetweenmiR-1298down-regulationandhigher io
DNA methylation levels at the upstream CpG sites, the 2000bp miR-1298bindingsitesintheCx43mRNA3′-UTRregionwerecon- vas
sequence (21900 to +100bp) encompassing the CpG sites was structedintoadual-luciferasereportvector.Inthepresenceofthe cre
inserted into a pGL3-basic plasmid, which was then transfected wild-typeCx43mRNA3′-UTR,miR-1298significantlydecreasedthe s/a
ipnltaosm2i9d3wTasceullssetdoacsotnhdeuccotnatlruocli.feInrdaseeeda,sstahye.AclnoneemdpftryagpmGeLn3t-bwaistihc rinelCatxiv4e3lumcRifNerAas3e′a-UctTivRityw(aFsigmuruet3aIt)e.dA,fttheertinhheibmitioRr-1y2e9ff8ecsteeodfmseiRqu-1e2n9c8e rticle-a
b
on the relative luciferase activity was abrogated (Figure 3J). Co- s
tfehreasweiladc-ttiyvpitey,sweqhuileentchee(CpGpGL3s-imteisR-o1f2t9h8e)fdraegmmoennsttrwateerdesmtreotnhgylalutecid- tluracnifsefreacsteioanctoivfitthye(Foigliugroew3Kit)h.TanheemrepstuyltvseccotonrfirdmidendotthaaltteCrxt4h3eirsealatdiivre- tract/1
bymethylase(mut-pGL3-miR-1298),theluciferaseactivitywasmarkedly 07
attenuated(Figure1K).Interestingly,treatingthemethylatedCpGsites ectFutarrtgheetrmofomrei,Rt-o12d9e8te.rminewhetherCx43isinvolvedinmiR-1298- /4/53
withmethyltransferaseinhibitor5-azasignificantlyrecoveredthelucifer- 4
inducedcellulareffects,weoverexpressedCx43inVSMCsusingalen- /3
aseactivity(Figure1K).ThisfindingsuggeststhatDNAmethylationoc- 1
tiviralvector(LV)(Cx43-LV,Figure4A).ThecoresequenceofCx43-LV 14
curring within the upstream DNA CpG sites affects miR-1298 7
onlycontainsCDS(codingsequence)ofCx43anddoesnotcontain 8
expression.WeconcludethatdecreasedmiR-1298expressioninASO b
arteriesisrelatedwiththeupstreamDNACpGsitemethylation. 3′-UTR,sotherearenomiR-1298bindingsitesinCx43-LV.Therefore, y g
u
miR-1298 could not inhibit Cx43 expression induced by Cx43-LV e
s
3.3 miR-1298 suppressed VSMC (Figure4A).AsshowninFigure4BandC,overexpressingCx43sig- t o
n
nificantlypromotedVSMCstoproliferateandmigrate.Intriguingly, 0
proliferation and migration 4
overexpressingCx43byCx43-LVsignificantlyattenuatedtheantipro- A
p
EthxecepsrsoivceesVsSoMfaCrtperrioolisfcelreartoiosins.4anTdompuigrrsauteiotnhearceelkluelyarceelflfuelcatrseovfednytssrein- glifeetrhaetriv,ethaensdeadnattiamiingdraictaotreytehfafetcCtxo4f3miisRa-1d2ir9e8c(tFtigaurgreet4oDf–mGiR).-T1a2k9e8natnod- ril 20
1
gulatedmiR-1298expressioninASO,weintroducedamiR-1298mimic wasinvolvedinmiR-1298-inducedcellulareffects. 9
or2′OME-miR-1298(miR-1298inhibitor)intoculturedVSMCs.Flow
cytometryanalysisrevealed that miR-1298significantlyattenuated 3.5 miR-1298-targeted Cx43 to affect
the PDGF-induced acceleration ofcell-cycle progressionfrom the
phenotype switching, gap junction
G0/G1phaseintotheSphase(Figure2AandB).However,annexin
intercellular communication and
V/propidiumiodidedualstainingassayshowedthatmiR-1298didnot
Cx43/ERK signalling pathway in VSMCs
increasethenumberofapoptoticcellsinVSMCs(Figure2CandD).
Figure2E–GshowthatthePDGF-stimulatedVSMCproliferationwas TheabilityofVSMCstoproliferateandmigrateiscloselyrelatedto
significantly inhibited by the miR-1298 mimic, as determined by theircellularphenotype.VSMCsexhibitdistinctphenotypes:contract-
CCK-8 and EdU assays. We found that miR-1298 significantly de- ileandsyntheticforms.ContractileVSMCsarepredominantinthenor-
creasedthePDGF-inducedmigrationofVSMCs(Figure2H–K,asde- malmedia,whereassyntheticVSMCsthatproliferatefaster,migrate
terminedbywoundclosureassayandtranswellassay).Incontrast, moreandhaveagreatersyntheticindexareintheneointima,suggest-
inhibitingmiR-1298bymiR-1298antagomirincreasedthecellnumber ingthattheyparticipateinthearteriosclerosisprocess.13PDGFisa
538 W.Huetal.
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Figure2 miR-1298inhibitsVSMCproliferationandmigration.(AandB)CellcycleanalysisusingflowcytometryshowedthatmiR-1298(50nmol/L)
decreasedthePDGF-induced(20mg/L)progressionofVSMCsintotheSphase.(CandD)miR-1298(50nmol/L)didnotsignificantlyincreasethelevelof
apoptosisinserum-starved(48h)VSMCs,asdeterminedbyannexinV/PIdualstaining.(EthroughG)miR-1298(50nmol/L)significantlydecreased
PDGF-induced(20mg/L)VSMCproliferation,asdeterminedbythecellcountingkit-8(CCK8)and5-ethynyl-2′-deoxyuridine(EdU)assays.(HandI),
miR-1298(50nmol/L)significantlydecreasedPDGF-induced(10mg/L)VSMCmigration,asdeterminedbythewoundclosureassay.(JandK)miR-1298
(50nmol/L)significantlydecreasedPDGF-induced(10mg/L)VSMCmigration,asdeterminedbythetranswellassay.(L)2′OME-miR-1298(miR-1298in-
hibitor,100nmol/L)increasedthenumberofcellsintheSphase.(M)2′OME-miR-1298(100nmol/L)moderatelyinducedVSMCproliferationasdeter-
minedbyCCK8.(N)2′OME-miR-1298(100nmol/L)moderatelyinducedVSMCmigrationasdeterminedbyatranswellassay.(O)2′OME-miR-1298
(100nmol/L)didnotsignificantlyincreasethelevelofapoptosisinserum-starved(48h)VSMCs.n¼4–6;scalebars¼100mm(J),200mm(H,F);
*P¼0.05,**P¼0.005,***P¼0.001.
miR-1298AffectsVSMCFunctionbyTargetingCx43 539
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Figure3 Cx43isatargetofmiR-1298inVSMCs.(A)TherearetwomiR-1298bindingsitesinthe3′-UTRregionofCx43.ThemiR-1298seedse-
quenceinthe3′-UTRofCx43mRNAwasmutated,andthe3′-UTRregionofCx43wasclonedintothepsiCHECKTMdual-luciferasevector.(BandC)
TheCx43(green)proteinlevelwasmuchhigherinASOarteriescomparedwithnormalarteries.Onthecontrary,themiR-1298(red)levelwassig-
nificantlylowerinASOarteriescomparedwithnormalarteriesasdeterminedbyFISHandIFC(n¼8forboth).(D)Cx43expressionwasup-regulated
inASOarteryVSMCs(n¼5)comparedwithnormalarteryVSMCs(n¼5),asdeterminedbywesternblot.(E)TheexpressionofmiR-1298wasfound
tobeinverselycorrelatedwiththeproteinexpressionofCx43innormalandASOarteries,asdeterminedbywesternblotandqRT-PCRanalysis(n¼5).
(FandG)Cx43proteinexpressionwassignificantlydown-regulatedbythemiR-1298mimic(50nmol/L)andwasmoderatelyup-regulatedby
2′OME-miR-1298(100nmol/L),asdeterminedbywesternblotanalysis(n¼6forboth).(H)ThemiR-1298mimic(50nmol/L)minimallydown-
regulatedtheexpressionofCx43mRNA,asdeterminedbyqRT-PCR(n¼6).(I–K)AmiR-1298mimic(50nmol/L)wasco-transfectedwiththe
psiCHECK-2-vectorcontainingeitherthewild-typeormutatedCx433′-UTRsequence.miR-1298significantlydecreasedtherelativeluciferaseactivity,
whilemutationofthemiR-1298seedsequenceabrogatedtheinhibitoryeffectofmiR-1298ontherelativeluciferaseactivity.Co-transfectionofthe
controloligowiththewild-typeCx433′-UTRsequenceoremptyvectordidnotsignificantlyaltertherelativeluciferaseactivity(n¼3).Scalebars¼200mm
(B);*P¼0.05,**P¼0.01,***P¼0.001.
540 W.Huetal.
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Figure4 Cx43isinvolvedinmiR-1298inducedcellulareffects.(A)OverexpressionofCx43inVSMCswasachievedusingalentivirusvector rdio
(Cx43-LV),asdeterminedbywesternblotanalysis;miR-1298couldnotinhibitCx43expressioninducedbyCx43-LV,whichlackedCx43mRNA va
3′-UTRandmiR-1298bidingsites.(BandC)OverexpressingCx43promotedVSMCproliferationandmigration,asdeterminedbyCCK8andtranswell sc
re
assays(n¼6).(DandE)OverexpressingCx43byCx43-LVsignificantlyattenuatedtheantiproliferativeeffectofmiR-1298asdeterminedbyEdUassay s
/a
(n¼6).(FandG),OverexpressingCx43byCx43-LVsignificantlyattenuatedtheantimigratoryeffectofmiR-1298asdeterminedbytranswellassay rtic
(n¼6).Scalebars¼200mm(DandF);*P¼0.05,**P¼0.01,***P¼0.001. le
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well-acknowledged stimulusthat induces phenotype changesfrom necessaryforthecontrolgrouptorecovertomaximumintensity.As t/1
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contractileVSMCstosyntheticVSMCs.14Interestingly,wefoundthat showninFigure5F,Cx43-LVup-regulatedtheGJICofVSMCs,whereas 7
/4
PDGF treatment also up-regulated Cx43 expression. As shown in miR-1298attenuatedthisprocess,asdeterminedbyFRAPusingsyn- /5
3
Figure5AandB,Cx43expressionwasup-regulatedinsyntheticVSMCs theticVSMCsthatparticipateinthearteriosclerosisprocess.13 4/3
1
after4daysofPDGFtreatmentasdeterminedbywesternblotanalysis Recentevidencesuggeststhat,inadditiontotheirwell-established 1
4
andIFC.IthasbeendescribedthatCx43down-regulationprevents function as channel-forming proteins, connexins might have func- 78
PDGF-induced phenotypic changes in smooth muscle cells and tionsindependentoftheirroleingapjunctions.19Thecytoplasmic by
g
up-regulates the contractile phenotypic markers SM-a-actin and C-terminusdomainofCx43interactswithnumeroussignallingcom- u
e
calponin.15,16Inourstudy,thePDGF-inducedup-regulationofCx43 plexessuchasb-catenin,PKCs,andsrc(Figure5G).Ithasbeenre- st o
wasdown-regulatedbymiR-1298overexpression(Figure5AandB), portedthatoverexpressionofCx43inosteoblastscanamplifyERK n 0
andthephenotypicmarkerchangesinthecontractileVSMCstoward andPKCdsignallingpathwayactivation.20Inthisstudy,overexpression 4 A
p
t(Fhiegusreyn5tCheatnidcDph).enotype was also partly prevented by miR-1298 otofCdox4w3ni-nreVgSuMlaCtesCalxs4o3amtopilnifiheibditERthKissfigunnacltliinogn,a(FnidgumreiR5-H12).98wasable ril 20
1
Cx43,theprincipalgapjunctionproteininVSMCs,servesasaninter- 9
cellularchannelthatregulatesthemovementofionsandothersignal- 3.6 Exogenous miR-1298 modulates Cx43
ling molecules.17 The fluorescence recovery after photobleaching
expression and inhibits vascular neointimal
(FRAP)assayisadirectandreliablemethodofmeasuringGJIC(Gap
formation in balloon-injured rat carotid
JunctionIntercellularCommunication).17IntheFRAPassay,recovery
arteries
offluorescenceinableachedcellistheresultoftheinfluxofnative
dye(5,6-CFDA)fromneighbouringcellsviagapjunctions(Figure5E). TodeterminetheeffectsofmiR-1298onVSMCsproliferationandneoin-
Therefore,theindexoffluorescencerecoveryisdirectlyproportional timalformationinvivo,weusedtheratcarotidballooninjurymodeland
tothedegreeoffunctionalgapjunctioncoupling.18Theanalysedfluor- LV to regulate miR-1298 or Cx43 expression in the vascular wall.
escencerecoveryindexisexpressedasR¼(I2I )/(I2I )×100%, miR-1298-LV and Cx43-LV could overexpress miR-1298 or Cx43;
t 0 0
whereI istheintensityofthephotobleachedfluorescence,Iisthein- miR-1298inhibitor-LVcouldgeneratestem–loopRNAwhichcould
0
tensityofpre-bleachedfluorescenceandI,inthisexperimentisthein- combineanddown-regulatemiR-1298.Vehiclecontrols(normalsaline),
t
tensityat3.5minafterphotobleaching,whichisthehalf-time(t ) empty vectorcontrols,andemptyvectorcontrolscontaininggreen
1/2
miR-1298AffectsVSMCFunctionbyTargetingCx43 541
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Figure5 miR-1298targetsCx43toaffectphenotypicchange,gapjunctionintercellularcommunicationandtheCx43/ERKsignallingpathwayin t o
n
VSMCs.(AandB)miR-1298down-regulatedCx43expressioninsyntheticVSMCstreatedbyPDGF(20mg/L)asdeterminedbyIFCandwestern 0
4
blotanalysis(n¼4to6).(CandD),miR-1298targetedCx43toaffectthephenotypemodulationofVSMCsandpartlyrestoredthecontractilepheno- A
p
typicmarkersSM-a-actinandcalponinasdeterminedbywesternblot(n¼4to6).(E)Representativephotomicrographs(×630)ofVSMCsundergoing ril 2
FRAP,withthetargetedcellindicated.Thefluorescenceofphotobleachedcellsrecoveredovertimeandstabilizedat7min.(F)TheGJICofsynthetic 0
1
VSMCstreatedwithPDGF(20mg/L)wassignificantlyup-regulatedbyCx43-LVanddown-regulatedbythemiR-1298mimic(50nmol/L),asdetermined 9
byFRAP(n¼6).(G)Eachconnexincontainsfourtransmembranedomains,twoextracellularloops,acytoplasmicloop,andcytosolicN-andC-termini.
ThecytoplasmicC-terminusdomainofCx43interactswithnumeroussignallingcomplexessuchassrc.(H)OverexpressionofCx43amplifiedERK
signalling,andmiR-1298suppressedthisfunctionofCx43(n¼6).Scalebars¼200mm(C)and30mm(AandE);*P¼0.05,**P¼0.01,***P¼0.001.
fluorescentprotein(GFP)mRNAwereincluded.TheLVsweredelivered 1298expressionlevelinvascularwallsignificantlydecreasedcompared
intothevascularwallaftertheballooninjuryasdescribedinSupplemen- withnormalvascularsamples,and miR-1298-LVre-overexpressed
tarymaterialonline.9Immunofluorescence(Figure6A)showedthatthe miR-1298 in injured vascular walls, as determined by qRT-PCR
LVs(green)weresuccessfullytransfectedintothevascularwall. (Figure6C).ISH(Figure6DandE)confirmedthatmiR-1298expression
In the balloon injury vascular, numerous VSMCs were found to levelsignificantlydecreasedafterballooninjury,andmiR-1298-LVre-
presentthroughoutthearterymediaandneointimaasdetermined overexpressedmiR-1298.Re-overexpressingmiR-1298significantlyin-
byIHC(Figure6B).Itwasfoundthatafterballooninjury,the miR- hibitedneointimalformationinratcarotidarteriesafterballooninjury;
542 W.Huetal.
incontrast,themiR-1298inhibitor-LVslightlyincreasedneointimalfor- ThetwomiR-1298bindingsitesinthe3′-UTRofCx43andthenega-
mation (Figure 6Fand G).Furtherinvestigationofthe proliferation tivecorrelationbetweenmiR-1298andCx43inASOandnormalvas-
markerKi67revealedthatmiR-1298-LVsignificantlyinhibitedVSMC culararteries in vivo suggest that Cx43 may be a miR-1298 target.
proliferationinvivo,asdeterminedbyIFC(Figure6HandI)andwestern Therefore,weexploredthepossibilitythatCx43maybeatargetof
blot(Figure6J)assays. miR-1298.WefirstvalidatedthatmiR-1298directlybindstheCx43
Furthermore,weinvestigatedwhetherCx43wasinvolvedinmiR-1298 3′-UTRregionstoregulateitsexpressionusingaluciferasereporteras-
inducedbiologicaleffectsinvivo.AsshowninFigure7AandB,balloonin- say.Then,usingCx43-LV,whichlacksmiR-1298bindingsites,wecon-
jurysignificantlyincreasedCx43expression,andwewereabletooverex- firmedthatmiR-1298couldnotinhibittheCx43expressioninducedby
press Cx43 using Cx43-LV in balloon-injured artery samples. The Cx43-LV.OverexpressingCx43byCx43-LVsignificantlyattenuated
up-regulationofCx43inballoon-injuredvascularwallswasattenuated theantiproliferativeandantimigratoryeffectofmiR-1298.Thesedata
byoverexpressingmiR-1298(Figure7C).However,miR-1298-LVcould suggestthatCx43isafunctionaldownstreamtargetofmiR-1298and
not inhibit Cx43 overexpression induced by Cx43-LV which lacked isinvolvedinmiR-1298-mediatedmodulationofVSMCfunction. D
Cx43mRNA3′-UTRandmiR-1298bidingsitesasdeterminedbywest- ItiswellestablishedthatVSMCproliferationandmigrationplayim- ow
n
ernblot(Figure7D).Furthermore,weinvestigatedtheroleofCx43in portantrolesinarteriosclerosis.6Inthecurrentstudy,wehavedemon- lo
a
d
VSMCproliferationinvivo.OverexpressingCx43byCx43-LVincreased stratedthatmiR-1298significantlyinhibitsVSMCproliferationand e
d
the level of the proliferation marker Ki67 and attenuated the migration.TheabilityofVSMCstoproliferateandmigrateiscloselyre- fro
miR-1298-LV-inducedinhibitionofKi67proteinexpressionasdeter- latedtotheircellularphenotype.VSMCsexhibitdistinctphenotypes: m
h
minedbywesternblot(Figure7E).AsshowninFigure7FandG,overex- contractileandsyntheticforms.ContractileVSMCsarepredominant ttp
s
pressing Cx43 by Cx43-LV, which lacks miR-1298 binding sites, innormalmedia,whereassyntheticVSMCs,whichproliferatefaster, ://a
attenuatedthemiR-1298-LV-inducedinhibitionofCx43expressionas migratemore,haveahighersyntheticindex,andarefoundintheneoin- ca
d
determinedbyIHC.AsshowninFigure7HandI,overexpressingCx43 tima,suggestingthattheyparticipateinarteriosclerosis.13Inourstudy, e
m
increasedneointimalformationandattenuatedthemiR-1298-LV-induced the PDGF-induced up-regulation of Cx43 was down-regulated by ic
.o
inhibitionofneointimalformation.Theseresultsdemonstratethatex- miR-1298overexpression,andthephenotypicmarkerchangesinthe u
p
ogenousmiR-1298inhibitedneointimalformationbytargetingCx43. contractileVSMCstowardthesyntheticphenotypewerealsopartly .co
m
preventedbymiR-1298. /c
a
4. Discussion Cx43,aprincipalgapjunctionproteininVSMCs,mediatestheinter- rd
cellularcommunicationbetweenVSMCsandplaysavitalroleinar- iov
a
Inthepreviousstudy,weusedamicroRNAqRT-PCRarraytoprofile teriosclerosis.17,24ThecontributionofCx43tothedevelopmentand sc
themicroRNAexpressionsignaturesinnormalhumanarteriesandhu- progressionofarteriosclerosishasbeenpreviouslystudiedinCx43+/2 res
manarterieswithASO.10WefoundthatmiR-1298isoneofthemost LDLR2/2 mice;25,26 down-regulation of Cx43 was shown to limit /artic
significantlydown-regulatedmicroRNAsinthearteriesofASOpa- neointimalformationafteravascularinjurybydecreasingtheinflamma- le
-a
tients.Inthisstudy,wedetectedthelocalizationofmiR-1298inhuman toryresponseandreducingmigration,proliferation,andphenotypic b
s
ASOarteriesandfoundthatmiR-1298exhibitstissuetropismandlo- switchingofVSMCs.PreviousstudieshaveaddressedthatCx43over- tra
c
calizesintheVSMCofthearterywall.Thesecharacteristicsindicate expression in VSMCs promotestheir proliferation, migration, and t/1
0
thatmiR-1298mayaffectVSMCfunctioninASO. phenotypicswitching.12,15,17,27HowdoesCx43workasaregulator 7
/4
MicroRNAgenesaremainlytranscribedbyRNApolymerase.They ofVSMCproliferationandmigration?Cx43mayactasintercellular /5
3
areinitiallytranscribedaslargeprecursors,calledprimarymicroRNAs channelsbyprovidingpathwaysforthedirectintercellularexchange 4/3
(pri-miRNAs).Pri-miRNAsarethenprocessedbytheRNase-IIIen- ofsecondarymessengers,ions,andsignallingmolecules.28Thissystem 11
4
zymeDroshaandDicertogeneratematuremiRNAproducts.How- ofcommunicationallowsVSMCstoquicklyspreadtheirfunctional 78
b
ever, the mechanism of miR-1298 dysregulation in ASO remains statetotheirneighboursviavariousstimulatingsignals,whichcanin- y
g
unknown.Atfirst,wedetectedthelevelofpri-miR-1298wassignifi- ducecellproliferationandmigration.Inthepresentstudy,usingthe u
e
s
cantlylowerinASOarteriescomparedwithnormalarteries.Thisfind- FRAPassay,wefoundthatmiR-1298mayinhibittheup-regulationof t o
ingindicatesthatmiR-1298istranscriptionallydown-regulatedinASO. Cx43andattenuateintercellularexchange,preventingadditionalnear- n 0
4
DNAmethylation-mediatedepigeneticsilencingofmicroRNAshas bycellsfrombecomingactivatedandleadingtotheinhibitionofcell A
p
pmreetvhioyluastliyonbeiseanmdaejmorocnasutrsaeteodfminicsreovReNraAlcdaynscreergsu,laantidonabinecrraanncterD.2N1,2A2 pthraotliafellorawtisosnoamnedemxtigrarcaetilolunla.rCsxu4b3stacnocuelds,asulscohaacstCaas2a+,hteomeincthearntnheel ril 20
1
However,onlyafewreportshaveaddressedDNAmethylationinath- cell.Thus,overexpressionofCx43increasesthelevelofintracellular 9
eroscleroticlesions,23andDNAmethylationaffectingindividualmicro- Ca2+.29 Ca2+is a highly versatile second messenger that controls
RNAinatherosclerosishasremainedpoorlystudied.Wethenanalysed VSMCcontraction,proliferation,andmigration.30Ontheotherhand,
theupstreamDNAfeaturesofmiR-1298corecodingsequenceand eachconnexincontainsfourtransmembranedomains,twoextracellu-
foundCpG-richfragment.Usingapyrosequencingassay,we found larloops,acytoplasmicloop,andcytosolicN-andC-termini.20Recent
themiR-1298geneupstreamDNACpGsiteswerehypermethylated evidencesuggeststhat,inadditiontotheirwell-establishedfunctionas
inASO.TreatingVSMCswithaDNAmethyltransferaseinhibitorsig- channel-formingproteins,connexinsmighthavefunctionsthatarein-
nificantlyincreasedthepri-miR-1298andmaturemiR-1298levels.Fur- dependentoftheirroleinjunctions.19ThecytoplasmicC-terminus
thermore,theluciferasereporterassayconfirmedthattheupstream ofCx43hasbeenimplicatedintheregulationofCx43functionbyin-
CpG-richfragmentofthemiR-1298coresequenceaffectsthetran- teractingwithnumeroussignallingcomplexes,suchasb-catenin,src,
scription of the downstream gene. This finding indicates that andPKCs.31Thesechannel-independenteffectsareinvolvedinthe
miR-1298down-regulationiscloselyrelatedwithhypermethylation controlofcellmigration,proliferation,andphenotypicchange.32,33In
oftheupstreamDNACpGsites. currentstudy,wehavefoundthatoverexpressionofCx43inVSMCs
miR-1298AffectsVSMCFunctionbyTargetingCx43 543
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Figure6 ExogenousmiR-1298inhibitsvascularneointimalformationandVSMCproliferationinballoon-injuredratcarotidarteries.(A)Fluorescent
dye(GFP)-labelledcontrololigonucleotides(green)showedthattheLVsweresuccessfullytransfectedintothecellsofthevascularwalls.(B)Inthe
ballooninjuryvascular,numerousVSMCswerefoundtopresentthroughoutthearterymediaandneointimaasdeterminedbyIHC.(C)qRT-PCRana-
lysisshowingthelevelofmiR-1298ineachgroup.Afterballooninjury,themiR-1298expressionlevelinthevascularwallsignificantlydecreasedcom-
paredwithnormalvascularsamples;miR-1298-LVre-overexpressedmiR-1298,andthemiR-1298inhibitor-LVslightlydown-regulatedmiR-1298in
injuredvascularwall.(DandE)FISHanalysisshowingthelevelofmiR-1298(red)ineachgroup.(FandG)IntheLV-transfectedratcarotidarteryballoon
injurymodel,thedegreeofneointimalformation(intima-mediaratio,I/M)wassignificantlydecreasedbyoverexpressingmiR-1298.(H–J)IFCandwest-
ernblotanalysesshowingtheexpressionofproliferationmarkerKi67ineachgroup,whichindicatedthatoverexpressingmiR-1298inhibitedVSMC
proliferation invivo.n¼6 to 10;Scale bars¼200mm (top of AandB, bottom of F),50mm(bottom of AandB, D, topofF,H);*P¼0.05,
**P¼0.005,***P¼0.001.