Article
TheLongNoncodingRNAlncTCF7PromotesSelf-
StemCellsthrough
Highlights
dThelongnoncodingRNAlncTCF7ishighlyexpressedinliver
cancertissuesandCSCs
dLncTCF7isimportantforself-renewalofliverCSCs
dLncTCF7activatestheWntsignalingpathwaythroughTCF7
Authors
YanyingWang,LeiHe,...,
RunshengChen,ZusenFan
Correspondence
InBrief
Wangetal.haveidentifiedalong
noncodingRNA,lncTCF7,thatactivates
Wntsignalingtopromotelivercancer
stemcellself-renewalandtumor
propagation.Targetingthispathway
Wangetal.,2015,CellStemCell16,413–425
April2,2015a2015ElsevierInc.
http://dx.doi.org/10.1016/j.stem.2015.03.003
dLncTCF7recruitstheSWI/SNFcomplextoactivatetheTCF7
promoter
expression
ActivationofWntSignaling
GraphicalAbstract
RenewalofHumanLiverCancer
couldhelpaddressthehighrecurrence
andheterogeneityoflivercancer.
AccessionNumbers
GSE66515
GSE66529
crs@sun5.ibp.ac.cn(R.C.),
fanz@moon.ibp.ac.cn(Z.F.)
lncTCF7
cs,
100853,
nalingprimesliverCSCself-renewalandtumorprop-
tial.LncRNAsfunctioninawiderangeofbiologicalprocesses
andcanregulategeneexpressionincisorintransbydiverse
HCCsubjectsdieeachyear.Althoughpreviousstudiesidentified
manyaberrantlyexpressedprotein-codinggenesinHCC,novel
molecularmarkersthatcanhelpinearlydiagnosisandrisk
2013;Lietal.,2011),suggestingthatmutationsofsomeindivid-
ualsubunitspromotetumorigenesis.Additionally,theSWI/SNF
complexhasbeenimplicatedinbeinginvolvedincontrolling
assessmentarestillurgentlyneeded(Jietal.,2009;Yamashita
andWang,2013).Newtherapeuticapproachesarelikelytoderive
fromanimprovedunderstandingofthemolecularbasisofHCC.
mammalianstemcellself-renewalanddifferentiation(Eroglu
etal.,2014;Yuetal.,2013;Zengetal.,2013).However,how
theSWI/SNFcomplexfunctionsinCSCsremainselusive.In
agation.Insum,therefore,wehaveidentifiedan
lncRNA-basedWntsignalingregulatorycircuitthat
promotestumorigenicactivityinlivercancerstem
cells,highlightingtherolethatlncRNAscanplayin
tumorgrowthandpropagation.
INTRODUCTION
Livercanceristhefifthmostcommonlydiagnosedcancerandthe
secondmostfrequentcauseofcancerdeathinmenworldwide
(Jemaletal.,2011).Hepatocellularcarcinoma(HCC)represents
themajorhistologicalsubtype,accountingforC2470%–85%of
casesofprimarylivercancer.Unfortunately,the5-yearsurvival
rateofHCCsubjectsremainspoor,andmorethan750,000
mechanisms(BatistaandChang,2013;CechandSteitz,
2014;UlitskyandBartel,2013).LncRNA-mediatedbiologyhas
beenimplicatedinmanycellularprocesses(UlitskyandBartel,
2013).Incancer,lncRNAshavebeenreportedtoactasaprom-
inentlayeroftranscriptionalregulation,oftenbycollaborating
withchromatinremodelingcomplexes(Guptaetal.,2010;Pre-
nsneretal.,2013;Yangetal.,2013;Yuanetal.,2014).The
SWI/SNFcomplexisanevolutionallyconservedmultisubunit
complexthatmobilizesnucleosomesandremodelschromatin
usingtheenergyofATPhydrolysis(Helmingetal.,2014;Wilson
andRoberts,2011).TheSWI/SNFcomplexassociateswithtran-
scriptionfactors,coactivators,orcoexpressorstoregulategene
expression(Tolstorukovetal.,2013;Youetal.,2013).Inactivat-
ingmutationsinseveralSWI/SNFsubunitshavebeenidentified
invarioushumancancers(Jonesetal.,2010;Kadochetal.,
OurdatasuggestthatlncTCF7-mediatedWntsig-
denylated,yetthisclassoftranscriptshaslimitedcodingpoten-
CellStemCell
Article
TheLongNoncodingRNA
Self-RenewalofHumanLiver
throughActivationofWntSignaling
YanyingWang,
1,5
LeiHe,
2,5
YingDu,
1
PingpingZhu,
1
Guanling
ChongLi,
1
PengyanXia,
1
GengZhang,
1
YongTian,
4
Runsheng
1
CASKeyLaboratoryofInfectionandImmunity,InstituteofBiophysi
2
DepartmentofHepatobiliarySurgery,PLAGeneralHospital,Beijing
3
UniversityofChineseAcademyofSciences,Beijing100049,China
4
CASKeyLaboratoryofRNABiology,InstituteofBiophysics,Chinese
5
Co-firstauthor
Correspondence:crs@sun5.ibp.ac.cn(R.C.),fanz@moon.ibp.ac.cn(Z.F.)
http://dx.doi.org/10.1016/j.stem.2015.03.003
SUMMARY
Hepatocellularcarcinoma(HCC)isthemostpreva-
lentsubtypeoflivercancer,anditischaracterized
byahighrateofrecurrenceandheterogeneity.Liver
cancerstemcells(CSCs)maywellcontributetoboth
ofthesepathologicalproperties,butthemecha-
nismsunderlyingtheirself-renewalandmaintenance
arepoorlyunderstood.Here,usingtranscriptome
microarrayanalysis,weidentifiedalongnoncoding
RNA(lncRNA)termedlncTCF7thatishighlyex-
pressedinHCCtumorsandliverCSCs.LncTCF7
isrequiredforliverCSCself-renewalandtumorprop-
agation.Mechanistically,lncTCF7recruitstheSWI/
SNFcomplextothepromoterofTCF7toregulate
itsexpression,leadingtoactivationofWntsignaling.
Promotes
CancerStemCells
Huang,
1,3
JianjunLuo,
4
XinlongYan,
1
BuqingYe,
1
Chen,
4,
andZusenFan
1,3,
ChineseAcademyofSciences,Beijing100101,China
China
AcademyofSciences,Beijing100101,China
Thehighrateofrecurrenceandheterogeneityarethetwo
majorfeaturesofHCC(Visvader,2011).Recentstudieshave
suggestedthatheterogeneityisaresultofthehierarchicalorga-
nizationoftumorcellsbyasubsetofcellswithstem/progenitor
cellfeaturesknownascancerstemcells(CSCs)(Easwaranetal.,
2014).TheseCSCswithintumorbulkdisplaythecapacitytoself-
renew,differentiate,andgiverisetoanewtumor(Visvaderand
Lindeman,2012),accountingforahierarchicalorganizationof
heterogeneouscancercellsandahighrateofcancerousrecur-
rence.LiverCSCscanbeenrichedwithseveraldefinedsurface
markers,includingepithelialcelladhesionmolecule(EpCAM),
CD133,CD13,CD90,CD44,CD24,andcalciumchannela2d1
subunit,amongothers(Haraguchietal.,2010;Leeetal.,2011;
Maetal.,2007;Zhaoetal.,2013).However,howliverCSCssus-
taintheirself-renewalremainslargelyunknown.
LongnoncodingRNAs(lncRNAs)aredefinedastranscripts
longerthan200nucleotides(nt)thatare5
0
cappedand3
0
polya-
CellStemCell16,413–425,April2,2015a2015ElsevierInc.413
thisstudy,wefoundthatlncTCF7isrequiredfortheself-renewal
maintenanceofliverCSCs.LncTCF7recruitstheSWI/SNFcom-
plextotriggerTCF7expression,leadingtoactivationofWnt
signalingforprimingliverCSCself-renewal.
RESULTS
LncTCF7IsHighlyExpressedinLiverCSCs
AlthoughCD13(Haraguchietal.,2010)andCD133(Maetal.,
2007)havebeenwidelyusedasliverCSCsurfacemarkers,
CD133orCD13alonecouldenrichmorecellpopulationsin
HCCcelllinesorHCCprimarysamples(FiguresS1AandS1B).
Haraguchietal.(2010)demonstratedthattheCD13
+
CD133
+
cellfractionexhibitedslowgrowthcomparedwiththeircounter-
partCD13
C0
CD133
C0
cells.Moreover,theCD13
+
CD133
+
cellfrac-
tionwashighlyresistanttochemicaldrugtreatmentscompared
withtheirCD13
C0
CD133
C0
subset.Notably,weobservedthatthe
CD13
+
CD133
+
cellsubsetsweresuccessfullyenrichedinseven
celllinesoutofnineHCCcelllineswetestedandinover90%of
30HCCprimarysamplesweexamined(FigureS1B).Thus,we
combinedCD13andCD133toenrichtheCD13
+
CD133
+
cell
fractionandtherebyidentifythemashepaticCSCsinthisstudy.
WethensortedCD13
+
CD133
+
cells,presumptivelyliver
CSCs,fromHep3B,Huh7,andPLC/PRF/5HCCcelllines.
Actually,theCD13
+
CD133
+
subsetderivedfromHep3Bcells
significantlyenhancedoncosphereformationcomparedto
CD13
C0
CD133
C0
cells(10.3%±1.3%versus2.6%±0.3%;p=
0.003)(FigureS1C).Inaddition,thetumorsphereformation
frequenciesofCD13
+
CD133
+
andCD13
+
CD133
C0
subsetsof
Hep3Bcellswere10.3%±1.3%versus6.1%±1.1%(p=
0.0253);CD13
+
CD133
+
versusCD13
C0
CD133
+
fractionsof
Hep3Bcellswere10.3%±1.3%to5.2%±1.3%(p=0.0226)
(FigureS1C).SimilarobservationswereobtainedusingHuh7
andPLC/PRF/5cells(FigureS1C).Consistently,thetumor-initi-
atingabilityoftheCD13
+
CD133
+
subsetderivedfromHep3B
cellswassignificantlyhigherthanthatofCD13
+
orCD133
+
cells
alone(FiguresS1DandS1E).Similarresultswereachievedwith
Huh7cells.Weconcludedthatthetumorigeniccapacityof
CD13
+
CD133
+
cellswasmuchhigherthanthatofCD13
+
or
CD133
+
cellsalone.Ofnote,therewasnosignificantdifference
intheproliferativecapacitybetweenCD13
+
CD133
+
cellsand
CD13
C0
CD133
C0
cellsthroughcell-cycleanalysis(FigureS1F).
ToidentifylncRNAsinvolvedinliverCSCs,weconductedtran-
scriptomemicroarrayanalysisofCD13
+
CD133
+
cells(hereafter
calledliverCSCs)andCD13
C0
CD133
C0
cells(hereafterreferred
toasnon-CSCs)sortedfromthethreeabovementionedHCC
celllines.286noncodingRNAtranscriptswereaberrantlyex-
pressedinliverCSCscomparedwithnon-CSCs(Figure1A),
withsomeofthosedifferentiallncRNAsvalidatedinliverCSCs
whenwedetectedtenrandomlyselectedlncRNAs(FigureS1G).
SincelncRNAscanactincistoregulateexpressionofneigh-
boringgenesorintransbydiversemechanisms(RinnandChang,
2012),weconcentratedonintergeniclncRNAs,whichwerehigh-
lyexpressedinliverCSCsandlocatedinthenearbystemtran-
scriptionfactorsandcodinggenesrelatedtostemsignaling
pathways.AmongthesehighlyexpressedintergeniclncRNAs,
wefocusedonanuncharacterizedlncRNA,termedlncTCF7
(genesymbolTCONS_00009511-XLOC_004555).LncTCF7is
oneofthemosthighlyexpressedlncRNAsinliverCSCs(Fig-
414CellStemCell16,413–425,April2,2015a2015ElsevierInc.
ure1AandFigureS1G),residingonchromosome5inhumansbe-
tweentheheatshock70kDaprotein4(HSPA4)andTcellfactor7
(TCF7)genes(Figure1B).LncTCF7wascomposedofthree
exonsandspannednearly3.6kilobases(kb),identifyingitasa
modestlyconservedlocus.WefurtherconfirmedthatlncTCF7
washighlyexpressedinliverCSCs(Figure1C),aswellasinonco-
spherecellsderivedfromHCCcelllinesandHCCprimarysam-
ples(Figures1Dand1E).Wenextexaminedacohortof37paired
HCCtumorandperi-tumortissues,alongwith14hepatitisBvirus
(HBV)-infectedlivercirrhotictissuesand8normallivertissues
(TableS1).WeobservedthatlncTCF7wassignificantlyhighlyex-
pressedinHCCtumors(Figure1FandFigureS1H)andHCCcell
lines(FigureS1I),whereasitwasalmostundetectableinliver
cirrhosissamplesandhealthylivertissues(Figure1FandFig-
ureS1H).Inaddition,lncTCF7wasnotdetectableinsomeother
normalprimarytissuesweexamined(FigureS1J),suggestinga
morerestrictedfashionoflncTCF7expression.
WefurtherexaminedthetranscriptsoflncTCF7inliverCSCs
bynorthernblot.OnlyonetranscriptvariantoflncTCF7wasin
liverCSCs,withalengthbetween500to1,000bases(Figure1G).
However,lncTCF7expressionwasmuchlowerinnon-CSCs.A
totallengthof683ntoflncTCF7transcriptwasdeterminedby
arapidamplificationofcDNAends(RACE)assay(FigureS1K),
whichdisplayednocodingpotentiality(FiguresS1LandS1M).
Furthermore,lncTCF7waslocalizedinthenucleiofHCCtumor
cellsthroughRNAfluorescenceinsituhybridization(RNA-
FISH)andcellularfractionationassays(Figures1Hand1I).
LncTCF7washighlyexpressedinoncospherecells,butitwas
poorlyexpressedinnon-spheretumorcells.Thesedataindicate
thatlncTCF7,asanlncRNA,washighlyexpressedinHCCtumor
tissuesandliverCSCs.
LncTCF7IsRequiredforSelf-RenewalMaintenanceof
LiverCSCs
TodeterminetheroleoflncTCF7inliverCSCself-renewal,we
silencedlncTCF7againsteitherexon1or3inliverCSCsusing
lentivirus-mediatedshorthairpinsRNAs(shRNAs)(Figure2A).
shRNA-2(againstexon3)achievedmoreeffectiveknockdown
efficiency.Notably,lncTCF7depletionsignificantlyreduced
theexpressionofpluripotenttranscriptionfactorsSox2,Nanog,
andOct4comparedwithscrambledcontrol(shCtrl)cells
(Figure2B).Bycontrast,lncTCF7knockdowndidnotaffectc-
Mycexpression.Actually,c-Mycwashighlyexpressedinboth
lncTCF7silencedandshCtrlliverCSCs,whoseexpression
wasnotsignificantlyalteredinloss-of-functionexperiments(Fig-
ure2B).Additionally,lncTCF7knockdownremarkablyimpaired
generationofthefractionofCD13
+
CD133
+
cells(CSCs)(Fig-
ure2C).Importantly,lncTCF7depletiondramaticallyreduced
primary(1st),secondary(2nd),andtertiary(3rd)oncospherefor-
mationofHCCcelllinesandHCCprimarycells(Figure2D).
Consistently,lncTCF7silencingalsoimpaired2ndand3rdon-
cosphereformationderivedfromtheirprimaryoncospherecells.
SimilarobservationswereobtainedinlncTCF7silencedPLC/
PRF/5cells(datanotshown).Consequently,lncTCF7depletion
inHCCprimarytumorcellssignificantlyreducedtheexpres-
sionofSox2andNanog(Figure2E).Moreimportantly,wecon-
ducted2ndand3rdgenerationxenografttumorgrowthforthe
CD13
+
CD133
+
cellsubsetsderivedfromHCCcelllinesandpri-
marysamples.WenoticedthatCD13
+
CD133
+
cellpopulations
werestillenrichedfromthe2ndand3rdgenerationxenografttu-
mors(datanotshown).TheCD13
+
CD133
+
cellpopulationsfrom
the2ndand3rdgenerationdisplayedstrongertumor-initiating
capacities(FigureS2A)andoncosphereformingabilities(Fig-
ureS2B),indicatingalong-termself-renewalcapacityofthe
CD13
+
CD133
+
cellpopulation.Moreover,Ki67signalswere
notapparentlyalteredinliverCSCscomparedwithnon-CSCs
(FigureS2C).However,theamountofSox2wassignificantly
increasedinliverCSCs,butnotinnon-CSCs.Bycontrast,
lncTCF7overexpressionremarkablyenhancedSox2signalsin
liverCSCs,whereasKi67wasnotalteredinlncTCF7-overex-
pressingCSCscomparedtoempty-vector-treatedcells(Fig-
Figure1.LncTCF7IsHighlyExpressedin
LiverCSCs
(A)Geometricmean-centered,hierarchicalcluster
heatmapfrommicroarraydata.286annotated
noncodingRNAs(p<0.05)wererepresentedin
liverCSC(CD13
+
CD133
+
)comparedwithnon-
CSC(CD13
C0
CD133
C0
)cellssortedfromHCCcell
ureS2D).Similarobservationswereobtainedinxenografttumors
(FigureS2E),suggestinglncTCF7-mediatedtumorigenicityin
livercancerismainlycausedbytheself-renewalcapacityof
CSCs.
WenextexploredtheroleoflncTCF7intumor-initiatingforma-
tion.WeperformedsubcutaneousinjectionofBALB/cnude
micewithstableknockdownoflncTCF7andshCtrlcells.
LncTCF7depletionresultedinamuchweakertumorpresence
comparedwithshCtrlprimarytumorcellsasassessedbya
limitingdilutionxenograftanalysis(Figure2F),suggestingthat
lncTCF7knockdownreducedtumorinitiatingcapacity.More-
over,lncTCF7depletionsignificantlysuppressedxenograft
lines,Hep3B,Huh7,andPLC/PRF/5(PLC).Black
arrowheaddenoteslncTCF7.
(B)SchematicannotationoflncTCF7genomiclo-
cusonchromosome5.Bluerectanglesrepresent
exons(upperpanel).Sequenceconservationwas
analyzedbyPhylopsoftware(lowerpanel).
(C)LncTCF7wasdetectedinliverCSCsandnon-
CSCssortedfromHCCcelllinesbyquantitative
real-timePCRanalysis.Relativegeneexpression
valueswerenormalizedtoendogenous18SrRNA
unlessnotedotherwiseinthisstudy.Resultsare
shownasmeans±SD.
(DandE)LncTCF7wasanalyzedinoncosphere
andnon-oncospherecellsderivedfromthreeHCC
celllinesandHCCprimarycells.Resultsare
shownasmeans±SD.
(F)LncTCF7wasdetectedinHCCtumortissues
pairedwithadjacenttumortissues(n=37),liver
cirrhosistissues(n=14),andnormallivertissues
(n=8).Datawerenormalizedtoendogenous18S
rRNAexpressionandnormallivertissueswere
assignedavalueof1.
(G)A363ntprobeoflncTCF7waslabeledfor
northernblotanalysis.RNAswereextractedfrom
sphereandnon-spherecellsderivedfromHCC
primaryspecimens.U6RNAwasusedasa
loadingcontrol.
(H)LncTCF7intracellularlocalizationwasvisual-
izedinHCConcosphereandnon-spherecellsby
RNA-FISHassays.Representativeimagesof
lncTCF7inHuh7oncosphere(Sphere)andnon-
oncospherecells(Non-sphere)areshown.DAPI,
4’,6-diamidino-2-phenylindole.Probes,lncTCF7.
Scalebar,10mm.
(I)FractionationofHCConcospherecellsfollowed
byquantitativereal-timePCR(leftpanel)and
fractionationcontrolsbyimmnoblotting(right
panel).U1RNAservedasapositivecontrolfor
nucleargeneexpression.EEA1,endosomeanti-
gen1;N,Nuclearfraction;C,Cytoplasmicfraction.
Dataareshownasmeans±SD.p<0.05,p<
0.01,andp<0.001bytwo-tailedStudent’st
test.Dataarerepresentativeofatleastthreein-
dependentexperiments.SeealsoFigureS1and
TableS1.
CellStemCell16,413–425,April2,2015a2015ElsevierInc.415
Figure2.LncTCF7IsRequiredfortheSelf-RenewalMaintenanceofLiverCSCs
(A)LncTCF7wassilencedinliverCSCsbytwoindependentshRNAstargetingeitherexon1or3.LncTCF7-silencedstablecelllineswereestablished.Dataare
shownasmeans±SD.
(B)PluripotenttranscriptionfactorswereanalyzedinlncTCF7-depletedcellsbyquantitativereal-timePCR(leftpenal)andwesternblot(rightpanel)analyses.
Quantitativereal-timePCRresultsareshownasmeans±SD.
(C)CD13
+
CD133
+
(CSC)subpopulationsweredetectedinlncTCF7-depletedcellsbyFACSanalysis.Resultsareshownasmeans±SD.
(D)LncTCF7depletioncausesadiminishedoncosphere-formingcapacityinHCCcelllinesandHCCprimarycells.Therightpanelrepresentsstatisticalresultsas
means±SD.Scalebar,100mm.
(E)PluripotencytranscriptswereanalyzedinlncTCF7-silencedHCCprimarycellsbyquantitativereal-timePCRanalysis.Dataareshownas
means±SD.
(F)LncTCF7-silencedorshCtrlHCCprimarytumorcellsweredilutedandsubcutaneouslyimplantedintoBALB/cnudemice.Tumorswereobservedover
4months.n=12foreachgroup.
(legendcontinuedonnextpage)
416CellStemCell16,413–425,April2,2015a2015ElsevierInc.
tumorgrowthandtumorigeniccellfrequency(Figures2Gand
2H).Weobservedxenograftgrowthfor4monthsanddefined
athresholdtumorsizefordetectabilityoftumorsas30–
40mm
3
.SimilarobservationswereachievedinlncTCF7-
silencedHCCcelllines(FiguresS2F–S2H).Overall,lncTCF7
silencingabrogatesthetumorigeniccapacityofliverCSCs.
(G)LncTCF7-silencedorshCtrlHCCprimarytumorcellsweresubcutaneously
shownasmeans±SD.n=12foreachgroup.
(H)TumorigeniccellfrequencyinlncTCF7-depletedandcontrolprimarytumo
software/elda/).#1,sample#1;#19,sample#19;CI,confidenceinterval.
p<0.05,p<0.01,andp<0.001bytwo-tailedStudent’sttest.Datarepresent
Figure3.LncTCF7OverexpressionEnhancesTumorigenicCapacityof
(A)LncTCF7-overexpressingHCCprimarytumorcellswereestablished.oeVec,overexpress
shownasmeans±SD.
(B)OverexpressionoflncTCF7(oelncTCF7)resultedinelevatedexpressionofpluripotent
westernblot(right)analyses.Quantitativereal-timePCRdataareshownasmeans
(C)LncTCF7overexpressionenhancesthecapacityofoncosphereformation.Scale
shownasmeans±SD.
(D)LncTCF7-overexpressing(oelncTCF7)oremptyvector(oeVec)cellsderivedfrom
BALB/cnudemice.Tumorswereobservedover4months.n=12foreachgroup.
(E)LncTCF7-overexpressingorvectorcontrol(oeVec)treatedprimarycellswere
growth.Resultsareshownasmeans±SD.n=10foreachgroup.
(F)TumorigeniccellfrequencyinlncTCF7-overexpressingandemptyvectorcontrol
edu.au/software/elda/).#1,sample#1;#19,sample#19;oe,overexpression;CI,
p<0.05,p<0.01,andp<0.001bytwo-tailedStudent’sttest.Datarepresent
LncTCF7OverexpressionEnhancesTumorigenic
CapacityofLiverCSCs
WenextoverexpressedlncTCF7inHCCprimarytumorcells
andcelllinesandestablishedlncTCF7stablyoverexpressing
celllines(Figure3AandFigureS3A).LncTCF7overexpression
dramaticallyincreasedexpressionofpluripotenttranscription
injectedintoBALB/cnudemiceforobservationoftumorgrowth.Resultsare
rcellswasanalyzedwithalimitingdilutionassay(http://bioinf.wehi.edu.au/
atleastthreeindependentexperiments.SeealsoFigureS2.
LiverCSCs
ionemptyvector;oelncTCF7,overexpressionoflncTCF7.Dataare
factorsinHCCcellsasassessedbyquantitativereal-timePCR(left)and
±SD.
bar,100mm.oe,overexpression.Therightpanelrepresentsstatisticalresults
HCCprimarytumorcellsweredilutedandsubcutaneouslyimplantedinto
subcutaneouslyinjectedintoBALB/cnudemiceforobservationoftumor
(oeVec)cellswasdeterminedwithlimitingdilutionassays(http://bioinf.wehi.
confidenceinterval.
atleastthreeindependentexperiments.SeealsoFigureS3.
CellStemCell16,413–425,April2,2015a2015ElsevierInc.417
(legendonnextpage)
418CellStemCell16,413–425,April2,2015a2015ElsevierInc.
factors(Figure3B).Notably,lncTCF7overexpressionsignifi-
cantlyenhancedoncosphereformationandtumor-initiating
capacity(Figures3Cand3D).Consequently,lncTCF7overex-
pressiondramaticallyaugmentedxenografttumorgrowth(Fig-
ure3E)andtumorigeniccellfrequency(Figure3F).Wetested
atleastsixHCCprimarysamplesandachievedsimilarobserva-
tions.SimilarresultswerealsoobservedinlncTCF7-overex-
pressedHCCcelllinessuchasHep3BandHuh7cells(Fig-
ureS3).Alltogether,thesedataindicatethatlncTCF7playsa
criticalroleintheself-renewalmaintenanceofliverCSCs.
LncTCF7TriggersTCF7ExpressiontoActivateWnt
Signaling
ToexplorethetargetgenesoflncTCF7,weestablishedlncTCF7-
upstreamofTCF7intheregulationofliverCSCself-renewal,we
rescuedTCF7expressioninlncTCF7-silencedHCCcells(Fig-
ure4E).Interestingly,TCF7restorationrescuedtheoncosphere
formationabilityreducedbylncTCF7depletion(Figure4F).
Additionally,TCF7restorationalsorescuedlncTCF7-depletion-
reducedtumor-initiatingcapacityandxenografttumorgrowth
(datanotshown).LncTCF7silencingdramaticallydownregu-
latedexpressionofsomemajorWntmolecules,including
Wnt7a,Wnt4,andWnt2b(Figures4Aand4B).Amongthese
threeWntmolecules,lncTCF7depletionreducedexpressionof
Wnt7athemost.TofurthervalidatewhetherWnt7aisadown-
streameffectoroflncTCF7,weincubatedlncTCF7-silenced
HCCprimaryCSCswithrecombinantWnt7aforasphere
formationassay.WefoundthatWnt7arescuedtheoncosphere
examined.
(right
peri-
silencedHCCprimaryCSCcellsandconductedtranscriptome
microarrayanalysis.LncTCF7knockdownresultedindifferential
expressionof2,491genes(FigureS4A),suggestingthatlncTCF7
regulatesmanygenesthatrelatetoimportantsignalingpro-
cessesincludingtheWntsignalingpathway(FigureS4B).
Notably,lncTCF7knockdownsignificantlyreduceditsnearby
protein-codinggeneTCF7andsomemajorWntsignalingtargets
(Figure4A).Bycontrast,HSPA4andtheotherneighboringgenes
displayednosignificantchanges.Wethenvalidatedtheseob-
servationsinHCCcelllines(Hep3B,Huh7,andPLC)orHCCpri-
marycells(atleastsixsamples).Consistently,lncTCF7silencing
remarkablyreducedtheexpressionofTCF7andmajorWnttar-
getsinHCCcelllinesandHCCprimarycellsweexamined(Fig-
ures4Band4CandFiguresS4CandS4D).Incontrast,lncTCF7
depletiondidnotaltertheexpressionofHSPA4andtheother
neighboringgenes(FiguresS4CandS4E).Inaddition,lncTCF7
overexpressioninHCCcelllinesorHCCprimarycellsremark-
ablyenhancedtheexpressionofTCF7andmajorWnttargets
(FigureS4F).ThesedatasuggestthatlncTCF7activatesthe
WntsignalingpathwayinHCC.
WefurthertestedtheexpressionlevelsoflncTCF7andTCF7
inHCCsubjectsbyquantitativereal-timePCR.Wenoticed
thatlncTCF7expressionwaspositivelycorrelatedwiththe
expressionofTCF7(Pearsoncorrelationcoefficientr=0.693,
p<0.001)(Figure4D).TodeterminewhetherlncTCF7functions
Figure4.LncTCF7TriggersTCF7ExpressiontoActivateWntSignaling
(A)Mean-centered,hierarchicalclusteringofgenesalteredinlncTCF7-silenced
(B)DifferentialgeneswerevalidatedinlncTCF7-silencedHCCcelllinesandHCC
silencedcelllines(Hep3B,Huh7,andPLC)andsixHCCprimaryspecimenswe
means±SD.
(C)TCF7andCCND2wereanalyzedbyimmunoblotting.
(D)ExpressionlevelsoflncTCF7andTCF7weredetectedin30HCCsample
expressionandshownasDCT(thresholdcycle).
(E)TCF7wasoverexpressedinlncTCF7-silencedHuh7cells.oe,overexpression.
(F)TCF7overexpressionrescuessphereformationabilityreducedbylncTCF7depletion
100mm.Percentagesofsphere-formingcellswerecalculatedasmeans±SD
(G)TCF7wasdetectedinHCCtumorandperi-tumortissues(n=30)bywestern
Quantitativereal-timePCRdatawerenormalizedto18SrRNAexpressionand
(H)TCF7wasvisualizedinHuh7oncosphereandnon-spherecellsbyimmunofluores
(I)TCF7wasdetectedinHCCsamplesbyimmunohistochemistrystaining.Scale
(J)TCF7wasanalyzedinlive(n=48)anddead(n=32)HCCsubjectsinaccordan
(K)Sox2wasdetectedinTCF7-depletedorshCtrlHCCcells.
(L)TCF7depletionreducestherateofoncosphereformationincellsderivedfrom
(MandN)TCF7depletionreducestumor-initiatingcapacity(M)andtumorigeniccell
au/software/elda/).CI,confidenceinterval.
p<0.05andp<0.01bytwo-tailedStudent’sttest.Dataarerepresentativeof
formationabilityreducedbylncTCF7depletion(FigureS4G).
Additionally,Wnt7aalsorestoredtheexpressionlevelsof
TCF7,Sox2,andNanoginlncTCF7-depletedHCCprimary
CSCs(FigureS4H).DKK1(Dickkopf-relatedprotein1)wasre-
portedtobeanextracellularinhibitorofWntsignaling(Cui
etal.,2013).ThepresenceofDKK1wasabletoabolishtheonco-
sphereformationabilityenabledbylncTCF7overexpression
(FigureS4I).Consistently,DKK1treatmentalsoimpededthe
expressionlevelsofTCF7,Sox2,andNanoginlncTCF7-overex-
pressingHCCprimaryCSCcells(FigureS4J).Thesedata
suggestthatlncTCF7sustainstheself-renewalofliverCSCsup-
streamofTCF7.
TofurtherexploretheclinicalimplicationsofTCF7inHCC,we
analyzedtheexpressionofTCF7inHCCtumorandperi-tumor
tissues.WeobservedthatTCF7washighlyexpressedinHCC
tumors(Figure4G).Additionally,TCF7wasmainlylocalizedin
thenucleiofHCConcospherecells,whereasitwasalmostunde-
tectableinnon-spherecells(Figure4H).HighexpressionofTCF7
inHCCsampleswasfurtherconfirmedbyimmunohistochem-
istrystaining(Figure4I).Importantly,highexpressionofTCF7
predictedalowerrateof5-yearsurvivalinmembersofacohort
analyzedbyHoshidaetal.(2009)(Figure4J).Next,wesilenced
TCF7inHCCcellsandestablishedstablysilencedcelllines
(Figure4K).Notably,TCF7depletionsuppressedSox2expres-
sion.WeobservedthatTCF7silencingdramaticallyimpaired
inLiverCSCs
orshCtrl-treatedHCCprimaryCSCcells.
primarysamples.SimilarobservationswereobtainedfromthreelncTCF7-
ResultsofHep3BandSample#1arerepresented.Dataareshownas
sandsubjectedtocorrelationanalysis.Datawerenormalizedto18SrRNA
.Representativesphereformationisshownontheleftpanel.Scalebar,
panel).
blot(upperpanel)andquantitativereal-timePCR(lowerpanel)analyses.
tumortissueswereassignedavalueof1.
cencestaining.Scalebar,10mm.
bar,100mm.
cewiththe5-yearfollowupprotocoldevisedinHoshidaetal.(2009)(n=80).
HCCcelllinesandHCCprimarycells.Dataareshownasmeans±SD.
frequency(N)asanalyzedbyalimitingdilutionassay(http://bioinf.wehi.edu.
atleastthreeseparateexperiments.SeealsoFigureS4.
CellStemCell16,413–425,April2,2015a2015ElsevierInc.419
oncosphereformation,tumorinitiatingcapacity,andxenograft
growthinHCCcelllinesandprimarycells(Figures4L–4N,and
datanotshown).Insum,lncTCF7initiatesTCF7expressionto
activatetheWntsignalingpathway,leadingtoprimingofliver
CSCself-renewalandtumorpropagation.
LncTCF7RecruitstheSWI/SNFComplex
LncRNAsareconsideredtoexerttheirfunctionsthroughRNA-
interactingproteinsthatregulategeneexpressionbyvarious
mechanisms(GeislerandColler,2013).Therefore,weperformed
anRNApull-downassaywithbiotin-labeledlncTCF7tosearch
forpotentiallncTCF7-associatedproteins.BRG1,BAF170,and
SNF5,threecoresubunitsoftheSWI/SNFcomplex,wereiden-
tifiedtobindlncTCF7inliverCSCs(Figures5Aand5BandFig-
uresS5A–S5C).TheinteractionoflncTCF7withthethreeSWI/
SNFcomponentswasfurthervalidatedbyRNAimmunoprecip-
itation(RIP)(Figure5CandFigureS5D).However,lncTCF7
depletiondidnotinfluenceexpressionlevelsofBAF170,
BRG1,andSNF5(Figure5D),suggestingthatlncTCF7wasnot
involvedinthepost-translationalregulationoftheSWI/SNF
complex.Moreover,lncTCF7colocalizedwithBAF170inthe
nucleiofHCConcospherecells,whereaslncTCF7waspoorly
expressedinthenucleiofnon-spherecells(Figure5E).These
dataindicatethatlncTCF7associateswiththeSWI/SNFcom-
plexinthenucleiofliverCSCs.
WenextconstructedaseriesoflncTCF7truncationstomapits
bindingfragmentwiththeSWI/SNFcomplex.Wefoundthatthe
3
0
-endfragmentoflncTCF7(nt468to683)wassufficienttobind
BAF170,BRG1,andSNF5(Figure5F).Astablestem-loopstruc-
tureofthe3
0
-endfragmentwaspredictedbyRNAfoldinganal-
ysis(FigureS5E).Thebindingofthe3
0
-endregionoflncTCF7
withBAF170wasfurtherconfirmedbyanRNAelectricalmobility
shiftassay(EMSA)(Figures5Gand5H).UnlabeledlncTCF7
probescompetitivelydisruptedthisbindingcapacity.Addition-
ally,lncTCF7showedhighbindingaffinity(dissociationconstant
[Kd]36.24nM)(Figure5I).ThesedataindicatethatlncTCF7
directlybindstotheSWI/SNFcomplexwithhighaffinity.
LncTCF7TriggersTCF7ExpressiontoActivateWnt
Signaling
GiventhattheSWI/SNFcomplexregulatesgenetranscriptionby
bindingtopromoterlociandrefoldingchromatin(Robertsand
Orkin,2004),wethendetectedwhetherlncTCF7influenced
BAF170occupancyofthepromoterlocusoftheTCF7gene.
Weanalyzeda3kblocusupstreamfromthetranscriptionstart
sites(TSSs)oftheTCF7gene.WefoundthatlncTCF7depletion
abrogatedthebindingcapacityofBAF170with(approximately)a
C01,160toC01,048bpsegmentofTCF7promoter(Figure6A),
suggestingthatthissegmentwasthebindingsiteforlncTCF7.
Throughluciferasereporterassays,weidentified(approxi-
mately)aC01,200toC01,100bpsegmentofTCF7promoter
asasufficientbindingsiteforBAF170(Figures6B–6D).Finally,
weobservedviaaDNaseIdigestionassaythatlncTCF7or
BAF170knockdownsignificantlyimpairedthechromatinacces-
sibilityoftheTCF7locus(Figure4E).Bycontrast,BAF170was
notrecruitedonthepromotersofthestempluripotentfactors,
asassessedthroughchromatinimmunoprecipitation(ChIP)an-
alyses(FiguresS6A–S6D),suggestingthatBAF170hadanindi-
recteffectontheexpressionofstempluripotentfactors.These
420CellStemCell16,413–425,April2,2015a2015ElsevierInc.
datasuggestthatlncTCF7recruitstheSWI/SNFcomplexto
theTCF7promoter,therebyleadingtoitsactivation.
Notably,theSWI/SNFcomplexwashighlyexpressedinHCC
tumorsandliverCSCs(Figure6Fanddatanotshown),suggest-
ingthattheSWI/SNFcomplexmaybeimplicatedintheregula-
tionofliverCSCself-renewal.Consequently,BAF170depletion
dramaticallyreducedexpressionofTCF7andNanog(Fig-
ure6G),aswellasWntdownstreamtargetssuchasSox2,
CCND1,andCCND2.Additionally,BAF170knockdownsignifi-
cantlyimpairedtheabilityofoncosphereformationderived
fromHCCcelllinesandHCCprimarycells(Figure6H).Finally,
BAF170knockdownalsoremarkablyreducedtumor-initiating
capacityandxenografttumorgrowth(Figures6I–6K).Similarre-
sultswereobservedbydepletingSNF5andBRG1(datanot
shown).Alltogether,ourdatashowthatlncTCF7triggers
TCF7expressionthroughrecruitmentoftheSWI/SNFcomplex,
leadingtoprimingoftheself-renewalofliverCSCsandtumor
initiation.
DISCUSSION
Recently,CSCshavebeenidentifiedinmanysolidtumors,
includingbreast,lung,liver,brain,colon,prostate,andbladder
cancers(Haraguchietal.,2010;O’Brienetal.,2007;Visvader
andLindeman,2012).CSCshavestemcharacteristicssuchas
self-renewalanddifferentiation.CSCsmayaccountforcancer
relapseandmetastasisduetotheirinvasiveanddrug-resistant
capacities(Guptaetal.,2009).Severalsurfacemarkershave
beenidentifiedinliverCSCs.However,detailsofliverCSC
biologyremainlargelyunknown.Inthisstudy,weisolatedthe
CD13
+
CD133
+
subpopulationofcellsfromHCCcelllinesand
HCCprimarycellsandidentifiedthemasliverCSCs.Through
transcriptomemicroarrayanalysis,weidentified286differen-
tiallyexpressednoncodingRNAsinliverCSCs.Amongthese
highlyexpressedlncRNAsinliverCSCs,wedefinedanlncRNA
termedlncTCF7thatplaysacriticalroleinprimingtheself-
renewalofliverCSCs.
LncRNAshavebeenreportedtoplaywidespreadrolesingene
regulationandothercellularprocesses(BatistaandChang,
2013;FlynnandChang,2014).LncRNAsexerttheirfunctions
viadiversemechanisms,includingcotranscriptionalregulation,
modulationofgeneexpression,scaffoldingofnuclearorcyto-
plasmiccomplexes,andpairingwithotherRNAs(Ulitskyand
Bartel,2013).Notably,accumulatingevidenceshowsthat
lncRNAsmodulategeneexpressionasepigeneticmodifiers(Ka-
nekoetal.,2014;Klattenhoffetal.,2013;Prensneretal.,2013;
Tsaietal.,2010;Zhuetal.,2013).HerewefoundthatlncTCF7
activatesTCF7expressionincisthroughrecruitmentofthe
SWI/SNFcomplex.TCF7expressiontriggersWntsignalingto
initiateself-renewalofliverCSCs.AlthoughlncTCF7isex-
pressedintheantisensedirection,itfailstoformanlncTCF7-
TCF7mRNAcomplexfortheregulationofTCF7expression.
TheWntsignalingplaysapivotalroleinself-renewalanddiffer-
entiationofCSCs(Hoffmeyeretal.,2012;Myantetal.,2013).
AberrantactivationofWntsignalingisimplicatedinlivercancer
andotherchronicliverdiseases(Huchetal.,2013;Wangetal.,
2013a).HereweshowthatdepletionofTCF7andWntdown-
streamtargetgenesimpairstheself-renewalofliverCSCsand
theirtumor-initiatingcapacity,suggestingthatTCF7-mediated
Figure5.LncTCF7AssociateswiththeSWI/SNFComplex
(A)Biotin-RNApull-downswereperformedwithnuclearextractsofoncospherecellsusingfull-lengthlncTCF7transcript(sense),antisense,andothertwo
lncTCF7introncontrols.Thiswasfollowedbymassspectrometry.Banda,BRG1;bandb,BAF170;bandc,SNF5.
(B)ThreecorecomponentsoftheSWI/SNFcomplexwereconfirmedbyimmunoblotting.b-actinwasusedasaloadingcontrol.
(C)TheinteractionoflncTCF7withBAF170,BRG1,andSNF5wasverifiedbyanRNAimmunoprecipitation(RIP)assay.Resultsareshownasmeans±SD.p<
0.01bytwo-tailedStudent’sttest.
(D)LncTCF7depletiondoesnotaltertheproteinlevelsofBAF170,BRG1,andSNF5.
(E)LncTCF7wasvisualizedbyRNA-FISH,andimmunofluorescencestainingofBAF170inHuh7oncosphereandnon-spherecellswasperformed.Scalebar,
10mm.
(F)MappinganalysisofSWI/SNF-complex-bindingdomainsoflncTCF7.Shownarethefollowing:schematicdiagramoflncTCF7full-lengthandtruncated
fragments(toppanel);invitrotranscribedbiotin-labeledRNA(middlepanel);andwesternblotofBAF170,BRG1,andSNF5inRNApull-downsamplesbydifferent
lncTCF7fragments(bottompanel).
(G)BAF170proteinandbiotin-labeledlncTCF7(468-683nt)probeswereincubatedforanEMSAassay.
(H)DifferentconcentrationsofBAF170proteinwereincubatedwithlncTCF7probes(5nM)andfollowedbyEMSAassaysasabove(G).
(I)BindingaffinityoflncTCF7withBAF170asdeterminedbyfiveindependentEMSAassays.Non-linearregressioncurvesweregeneratedbyGraphPadPrism.
SeealsoFigureS5.
CellStemCell16,413–425,April2,2015a2015ElsevierInc.421
Figure6.LncTCF7TriggersTCF7ExpressiontoActivateWntSignaling
(A)Anapproximately1,160–1,048bpfragmentoftheTCF7promoterissufficientforthebindingofBAF170byChIP-quantitativePCRanalysis.Dataareshownas
means±SD.
(B)BAF170wassilencedinliverCSCsbytwoindependentshRNAs.
(legendcontinuedonnextpage)
422CellStemCell16,413–425,April2,2015a2015ElsevierInc.
WntsignalingiscriticalforsustainingliverCSCself-renewaland
tumorigenicity.
CSCsharborthestemcellpropertiesofself-renewalanddif-
ferentiation(VisvaderandLindeman,2012).Inthisstudy,we
foundthattherewasnosignificantdifferenceintheproliferative
capacitybetweenliverCSCsandnon-CSCs,asassessedby
cell-cycleanalysisandimmunofluorescencestaining.Actually,
CSCsexistmainlyintheG1/G0phaseorG0phasewithdormant
orslow-growingproperties(Haraguchietal.,2010).Assuch,it
mightbehardtodistinguishtheproliferationdifferencesbe-
whichincludescis-regulatoryelementsandtransactingfactors.
Amongalltransactingfactors,chromatinremodelingregulation
playsapivotalroleingenetranscription(Krastevaetal.,
2012;Narlikaretal.,2013),whichdependsonATP-dependent
chromatinremodelingcomplexes.TheSWI/SNFcomplexis
composedof12–15subunitscontainingoneofthetwocata-
lyticATPasesubunits,SMARCA4/BRG1orSMARCA2/BRM,
andseveralcorecomponentssuchasSMARCB1/SNF5,
BAF170,andBAF155(Helmingetal.,2014;WilsonandRob-
erts,2011).TheSWI/SNFcomplexregulatesgeneexpression
and
tweenliverCSCsandnon-CSCsviaKi67stainingorDNAcon-
tentsassayedbypropidiumiodide(PI).Also,theself-renewal
propertiesofdifferentcancertypesmayhavedifferentprolifera-
tioncharacteristics.Themaintenanceofself-renewalisan
extremelycomplicatedbiologicalprocess,whichiscontrolled
byepigeneticstates,pluoripotenttranscriptionfactors,epige-
neticcomplexes,andmanydevelopmentalpathways(Bou-
mahdietal.,2014;Ohnishietal.,2014;VisvaderandLindeman,
2012).OurdatasupporttheideathatlncTCF7-mediatedWnt
signalingactivationinhepaticCSCsplaysacriticalroleinthe
regulationofliverCSCself-renewal.
TCF7,asaTcellfactor,isbestknownforitsfunctioninT
lymphocytedevelopmentandmultipotentialhematopoietic
cellself-renewal(Weberetal.,2011).TCF7caninitiatethe
Wntsignalingcascadeasanupstreamtrigger(Cuietal.,
2013;Weberetal.,2011).TCF7
C0/C0
micedevelopedintestinal
andmammaryadenomas,implyingaroleforTCF7intumor
suppression(Yuetal.,2012).Inthisstudy,wedemonstrate
thatTCF7isrequiredfortheself-renewalofliverCSCsandtu-
morpropagationandactsasatumorpromoter.TCF7isregu-
latedbylncTCF7inliverCSCstotriggertheWntsignaling
pathway,whichprimestheself-renewalofliverCSCs.A
recentstudyreportedthatTCF7functionsasatumorenhancer
intumorigenesisafteroculartransplantationofembryonic
stemcell-derivedretinalprogenitorcells(Cuietal.,2013).
Consistently,ourfindingsrevealthatlncTCF7mediatedTCF7
expression,whichinitiatestheWntsignalingcascadetoprime
self-renewalofliverCSCsandhepatictumorigenicityasa
tumorpromoter.CSCsareanalogoustotissue-specificstem
cellsinthattheycontributetolong-termgrowthandare
responsibleforthemaintenanceandgrowthoftumors
(BeckandBlanpain,2013).Transcriptionalregulationis
oneofthekeyregulatorymechanismsdeterminingcellfate,
(CandD)DifferentlociofTCF7promoterwereconstructedintopGL3vector
shownasmeans±SD.
(E)LncTCF7orBAF170depletionincreaseschromatinaccessibilityatthepromot
(F)BAF170andBRG1werehighlyexpressedinHCCtumortissues(n=30).Data
wereassignedavalueof1.
(G)TCF7,pluripotenttranscripts,andWntpathwaytargetsweredetectedinBAF170-
blot(rightpanel)analyses.QuantitativePCRdataareshownasmeans±SD.
(H)BAF170depletionimpairsthecapacityofoncosphereformationincellsderived
sphere-formingcellswerecalculatedasmeans±SD(rightpanel).
(I)BAF170-silencedorshCtrlHCCprimarytumorcellsorHuh7cellswerediluted
during4months.n=6foreachgroup.
(J)BAF170-silencedorshCtrlHCCprimarytumorcellsandHuh7cellsweresubcu
Resultsareshownasmeans±SD.n=10foreachgroup.
(K)TumorigeniccellfrequencyinBAF170-silencedandshCtrlprimarytumorcells
elda/).#6,sample#6;CI,confidenceinterval.
p<0.05andp<0.01bytwo-tailedStudent’sttest.Datarepresentatleastthree
throughmobilizingnucleosomesandremodelingchromatin
usingtheenergyofATPhydrolysis,suggestingitscriticalrole
ingeneexpression.InactivatingmutationsinseveralSWI/SNF
subunitscausevarioushumancancers(Helmingetal.,2014;
Jonesetal.,2010;Kadochetal.,2013),suggestingthat
somecomponentsactastumorsuppressors.Arecentstudy
reportedthatthelongnoncodingRNASChLAP1associates
withtheSNF5subunittopromoteprostatecancerprogress
byantagonizingthefunctionoftheSWI/SNFcomplex(Pre-
nsneretal.,2013),whichimpliesthatSNF5functionsasatu-
morsuppressor.HerewefoundthatlncTCF7canrecruitthe
SWI/SNFcomplextotriggerTCF7expression,leadingtoprim-
ingofliverCSCself-renewal.Additionally,weobservedthat
BAF170,BRG1,andSNF5arehighlyexpressedinHCCspec-
imensandliverCSCs,whosedepletionimpairstumor-initiating
capacityandtumorpropagation(Figure6,anddatanotshown),
suggestingthatthesethreecomponentsactastumorpro-
moters.Therefore,howtheSWI/SNFcomplexcomponents
functionintumorigenesisremainstobefurtherinvestigated.
Inconclusion,lncTCF7canpromoteliverCSCself-renewal
andtumorpropagationthroughactivationofWntsignalingbyre-
cruitingtheSWI/SNFcomplextotheTCF7promoter.Ourfind-
ingsrevealthatlncRNAsmayrepresentanadditionallayerof
regulationofoncogenesis.
EXPERIMENTALPROCEDURES
NorthernBlotAnalysis
TotalRNA,extractedasdescribed(Wangetal.,2013b)fromhumanCSC
oncospheresandcontrolcells,wassubjectedtonorthernblotassays.An
lncTCF7-specificDNAsequence(nt320–683)wasclonedintopcDNA4/myc-
hisBVector.RadioactiveRNAprobewithalengthof363ntwasgeneratedus-
ing[a-
32
P]CTP(PerkinElmer)andtheRiboprobeinvitrotranscriptionlabeling
system(Promega).
subjectedtoluciferasereporterassaysinBAF170-silencedcells.Dataare
erofTCF7byDNaseIdigestionassays.Dataareshownasmeans±SD.
werenormalizedtoendogenous18SrRNAexpressionandperi-tumortissues
silencedHCCcellsbyquantitativereal-timePCR(leftpanel)andwestern
fromHCCcelllinesandHCCprimarycells.Scalebar,100mm.Percentagesof
andsubcutaneouslyimplantedintoBALB/cnudemice.Tumorswereobserved
taneouslyinjectedintoBALB/cnudemiceforobservationoftumorgrowth.
wasanalyzedwithalimitingdilutionassay(http://bioinf.wehi.edu.au/software/
independentexperiments.SeealsoFigureS6.
CellStemCell16,413–425,April2,2015a2015ElsevierInc.423
RNA-FISH
Fluorescence-conjugatedlncTCF7probeswereusedforRNA-FISH.
RNA-FISHwasperformedaspreviouslydescribed(Wangetal.,2014).
HybridizationwascarriedoutusingDNAprobesets(BiosearchTechnolo-
gies)accordingtotheprotocolofBiosearchTechnologies.Oncosphere
andcontrolcellswereobservedwithaFV1000confocallasermicroscope
(Olympus).
RNAPull-downandMassSpectrometryAssay
RNApull-downwasperformedasdescribed(Klattenhoffetal.,2013).Invitro
biotin-labeledRNAs(lncTCF7,itsantisenseRNA,andtwocontrolRNAsfrom
twointronsoflncTCF7)weretranscribedwiththebiotinRNAlabelingmix
(Roche)andT7RNApolymerase(Roche)treatedwithRNase-freeDNaseI
(Promega)andpurifiedwithRNeasyMiniKit(QIAGEN).BiotinylatedRNA
wasincubatedwithHCConcospherecellnuclearextracts,andpull-down
proteinswererunonSDS-PAGEgelsasdescribed(Xiaetal.,2013).Mass
spectrometryfollowed.
RNA-EMSAAssay
EMSAexperimentswereperformedusingaLightShiftChemiluminescent
RNAEMSAKit(ThermoScientific).Theshiftedsignalswerequantifiedand
plottedagainsttheamountofBAF170proteinwithGraphPadPrism6
(GraphPad).
StatisticalAnalysis
Datawereanalyzedwithadouble-sidedStudent’sttestusingtheSPSS13.0
softwareandGraphPadPrism6.Tumorigeniccellfrequencywascalculated
basedonextremelimitingdilutionanalysis(ELDA)(http://bioinf.wehi.edu.au/
software/elda/).p<0.05wasconsideredstatisticallysignificant.Forother
methodsseetheSupplementalInformation.
ACCESSIONNUMBERS
MicroarraydatahavebeendepositedintheNCBIGEOunderaccession
numbersGSE66515andGSE66529.
SUPPLEMENTALINFORMATION
SupplementalInformationforthisarticleincludessixfigures,onetable,and
SupplementalExperimentalProceduresandcanbefoundwiththisarticleon-
lineathttp://dx.doi.org/10.1016/j.stem.2015.03.003.
AUTHORCONTRIBUTIONS
Y.W.designedandperformedexperiments,analyzeddata,andwrotethepa-
per,andL.H.providedclinicalspecimensandanalyzeddata.P.Z.,G.H.,and
J.L.constructedplasmidsandanalyzeddata;Y.D.,X.Y.,C.L.,P.X.,andG.Z.
performedsomeexperiments;B.Y.andY.T.performedsomeexperiments
andanalyzeddata;R.C.initiatedthestudy;andZ.F.initiatedthestudyand
organized,designed,andwrotethepaper.
ACKNOWLEDGMENTS
WethankDr.ZeguangHan(ShanghaiJiaotongUniversitySchoolofMedicine,
Shanghai)forprovidinghepatocellularcarcinomacelllines.WethankDrs.
XinluWang,ZhenshengXie,JunyingJia,YanTeng,andJingLi(Cnkingbio
CompanyLtd,Beijing,China)fortechnicalsupport.Thisworkwassupported
bytheNationalNaturalScienceFoundationofChina(81330047,91419308,
81402459,81272270,and81101531);theStateProjectsofEssentialDrug
Researchanddevelopment(2012ZX09103301-041);973Programofthe
MOSTofChina(2015CB553705and2010CB911902);andtheStrategicPrior-
ityResearchProgramsoftheChineseAcademyofSciences(XDA01010407).
Received:November19,2014
Revised:February15,2015
Accepted:March7,2015
Published:April2,2015
424CellStemCell16,413–425,April2,2015a2015ElsevierInc.
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