HepatitisBVirusRegulatesApoptosisandTumorigenesisthroughthe
MicroRNA-15a-Smad7-TransformingGrowthFactorBetaPathway
NingningLiu,
a
TongJiao,
a,b
YanHuang,
a,b
WenjunLiu,
a
ZhiweiLi,
c
XinYe
a
CenterforMolecularImmunology,CASKeyLaboratoryofPathogenicMicrobiologyandImmunology,InstituteofMicrobiology,ChineseAcademyofSciences,Beijing,
China
a
;GraduateUniversityofChineseAcademyofSciences,Beijing,China
b
;302HospitalofPLA,Beijing,China
c
ABSTRACT
HepatitisBvirus(HBV)infectioncauseschronichepatitisinhundredsofmillionsofpeopleworldwide,whichcaneventually
leadtohepatocellularcarcinoma(HCC).Previously,wefoundthatHBVmRNAscanabsorbmicroRNA-15a(miR-15a)toaffect
apoptosisthroughtheBcl-2pathway.WeaskedwhetherHBVcouldinhibitapoptosisandpromotetumorigenesisthroughdif-
ferentpathways.Inthisstudy,wefoundthatthetransforminggrowthfactorH9252(TGF-H9252)pathway-inhibitoryfactorSmad7isa
noveltargetofmiR-15a.WedemonstratedthatHBVcanupregulatethelevelofSmad7bydownregulatingmiR-15a.Further-
more,weexaminedthelevelofSmad7inliversamplesfromHBV-infectedHCCpatientsandfoundthatHBVmRNAsareposi-
tivelycorrelatedwiththelevelofSmad7.Bytakingtheapproachofusingimmunoblottingandluciferasereporterassays,were-
vealedthatHBVcanabrogateTGF-H9252signalingviaupregulatingSmad7.ByusingannexinVstainingandcaspase3/7activity
assays,wefoundthatHBVcaninhibitTGF-H9252-inducedapoptosisofHepG2cells.WealsoshowedthatHBVcanpromotetumor
growthinBALB/cnudemicethroughupregulatingtheexpressionofSmad7.Inconclusion,wedemonstratedthatHBVcanup-
regulateSmad7expressionandinhibitTGF-H9252signaling,whichmakesthecellsresistanttoTGF-H9252-inducedapoptosisandpro-
motestumorigenesis.
IMPORTANCE
HepatitisBvirus(HBV)infectioncauseschronichepatitis,whichcaneventuallyleadtohepatocellularcarcinoma(HCC).TGF-H9252
signalingiscloselylinkedtoliverfibrosis,cirrhosis,andsubsequentHCCprogressionandplaysauniqueroleinthepathogene-
sisofHCC.Attheearlystageoftumorformation,TGF-H9252functionsasatumorsuppressorthatinhibitscellproliferationand
inducesapoptosis.Previously,wefoundthatHBVmRNAscanspongeoffmiR-15atoaffectapoptosisthroughtheBcl-2path-
way.Inthisstudy,weidentifiedthattheTGF-H9252-inhibitoryfactorSmad7isanoveltargetofmiR-15a.WerevealthatHBVcan
abrogateTGF-H9252signalingviaupregulatingSmad7,inhibitTGF-H9252-inducedapoptosis,aswellaspromotetumordevelopment.
OurstudyprovidesevidencetosupporttheideathatviralRNAscanexerttheirfunctionsascompetingendogenousRNAs
(ceRNAs)towardmicroRNAandparticipateinimportantcellularprocesses.
H
epatitisBvirus(HBV)infectionremainsamajorpublic
healthconcern,withH11022350millionpeoplebeingchronically
infectedworldwide(1).ChronicHBVinfectionisrelatedtothe
occurrenceanddevelopmentofhepatocellularcarcinoma(HCC),
whichisthethirdleadingcauseofcancermortality(2–4).
HBVisanenveloped,partiallydouble-strandedDNAvirus
withagenomesizeof3.2kb,anditreplicatesthroughanRNA
intermediateform(pre-C/C,pre-S,S,andXmRNAs)byreverse
transcription.ThemRNAsofHBVencodeseveralviralproteins,
includingthepolymerase,core,HBe,pre-S1,S2,S,andXproteins
(5).HBVinfectionhasbeenreportedtoplayanimportantrolein
regulatinghepatocyteapoptosisforpersistentsurvival(6–8).
MicroRNAs(miRNAs)aresmall,noncoding,single-stranded
RNAmoleculesthatareinvolvedintheregulationoftargetgene
expressioninmultiplecellularprocesses.Ithasbeenreportedthat
HBxpromotestumorigenesisbydownregulatingmicroRNA-148a
(miR-148a)(9)andinducesaberrantDNAmethylationbydown-
regulatingmiR-101(10).Also,HBVcanpromotecellproliferation
andtumorformationbydownregulatingmiR-122(11).Previously,
wefoundthatHBVinhibitsapoptosisbydirectlyspongingmiR-
15aandupregulatingBcl-2expression(12).Ithasbeenreported
thatmiR-15acanenhanceprostatecancerprogressionandpro-
motecellgrowthandsurvivalbytargetingBcl-2,CCND1,and
WNT3A(13).Inthisreport,weidentifiedthatmiR-15acanreg-
ulatethelevelofSmad7mRNAandenhancethetransforming
growthfactorH92521(TGF-H92521)signalingpathway.
TGF-H92521notonlyinhibitscellproliferationbutalsoinduces
apoptosisinhepatocytes,myeloidcells,andepithelialcells(14).
Smadproteinshavebeenidentifiedaskeysignaltransducersin
TGF-H92521-dependentgrowthinhibition(15).Whenactivatedby
TGF-H9252,theTGF-H9252typeIreceptorphosphorylatesSmad2,which
leadstotheassociationofSmad2withSmad4.Smad2/Smad4then
translocatestothenucleustopromotedownstreamgenetran-
scription.Smad7actsasaninhibitorofTGF-H9252signalingbyinter-
actingwiththeTGF-H9252typeIreceptortopreventthephosphory-
lationandactivationofSmad2(16).Ithasbeenreportedthat
Received26September2014Accepted11December2014
Acceptedmanuscriptpostedonline24December2014
CitationLiuN,JiaoT,HuangY,LiuW,LiZ,YeX.2015.HepatitisBvirusregulates
apoptosisandtumorigenesisthroughthemicroRNA-15a-Smad7-transforming
growthfactorbetapathway.JVirol89:2739–2749.doi:10.1128/JVI.02784-14.
Editor:J.-H.J.Ou
AddresscorrespondencetoZhiweiLi,lzw302@126.com,orXinYe,yex@im.ac.cn.
Copyright?2015,AmericanSocietyforMicrobiology.AllRightsReserved.
doi:10.1128/JVI.02784-14
March2015Volume89Number5jvi.asm.org2739JournalofVirology
onApril23,2017byguest
http://jvi.asm.org/
Downloadedfrom
Smad7canblockTGF-H9252-inducedgrowthinhibitionandinhibit
theapoptosisofFETcells,whichmayenhancethetumorigenicity
ofFETcells(14).
TGF-H9252signalingiscloselylinkedtoliverfibrosis,cirrhosis,and
subsequentHCCprogressionandplaysauniqueroleinthe
pathogenesisofHCC(17).Attheearlystageoftumorformation,
TGF-H9252functionsasatumorsuppressorthatinhibitscellprolifer-
ationandinducesapoptosis,whileatthelaterstageoftumor
progression,tumorcellslosetheirresponsetoTGF-H9252(18,19).
However,theexactfunctionofTGF-H9252signalinginchronicHBV
infectionandHBV-relatedHCCisstillunclear.Inthisstudy,we
identifiedthatSmad7,asaninhibitoryfactoroftheTGF-H9252path-
way,isanoveltargetofmiR-15a.Wefurtherdemonstratethat
HBVmRNAscaninterferewithTGF-H9252signalingthroughupregu-
latingSmad7expressionbyeliminatingmiR-15a,inhibitTGF-H9252-
inducedapoptosis,andfacilitatetumorformation.
MATERIALSANDMETHODS
Patientsandhumanspecimens.Livertissuesfrom40patientswithHCC
werecollectedfromthe302HospitalofPLA.Thepatientswerehospital-
izedduringJune2012toJuly2013.Theclinicalcharacteristicsofenrolled
subjectswerelistedinapreviousstudy(12).Writteninformedconsent
wasprovidedbyallstudyparticipants.Patientsampleswereassignedar-
bitraryidentificationnumbersbasedontheorderofenrollmentinour
study.Thestudyprotocolwasapprovedbytheethicscommitteeofthe
302HospitalofPLA.
HBVtransgenicmice.HBVtransgenicBALB/cmicewerepurchased
fromtheTransgenicEngineeringLab,InfectiousDiseaseCenter(Guang-
zhou,China),andweredescribedinourpreviousstudy(12).
Plasmidconstructs.TheSmad73=untranslatedregion(UTR)(nu-
cleotides[nt]1608to1627)(GenBankaccessionno.NM_001190821)was
amplifiedbyPCR,andthePCRproductwasclonedintothedual-lucifer-
asereporterpmirGLOvector(Promega,USA)andnamedpGLO-Smad7-
3=UTR.pGLO-Smad7-3=UTR-mut,withmutationsintheseedregionof
themiR-15a-bindingsite,wasgeneratedbysite-directedmutagenesis.
p3TP-lucwaspurchasedfromAddgene(plasmid11767)(20).Smad7and
Smad2wereclonedintopFLAG-CMV2(Sigma,USA)andpCMV-Myc
(Clontech,USA),respectively.pHBV1.3,carryingthefull-lengthHBV
genome,anditsmutantpHBV1.3-mut,whichcannotabsorbmiR-15a,
weregeneratedasdescribedpreviously(12).
Reagentsandantibodies.ThechemicallysynthesizedmiR-15ainhib-
itor,mimic,andnonspecificcontrolswerepurchasedfromRiboBioCo.,
Ltd.(Guangzhou,China).Thefollowingreagentsandantibodieswere
obtainedasindicated:therabbitanti-Smad7antibodieswereproducedby
immunizingrabbitswithHis-Smad7(aminoacids[aa]1to200)orHis-
Smad7(aa201to426),andtherabbitanti-humanpoly(ADP-ribose)
polymerase(PARP)antibody(CellSignaling,USA),theanti-FLAGanti-
body(Sigma,USA),theanti-Mycantibody(SantaCruz,USA),therabbit
anti-humanSmad2/3antibody(SantaCruz,USA),thephosphor-Smad2
(Ser465/467)rabbitmonoclonalantibody(MAb)(SantaCruz,USA),the
goatanti-humanlaminBantibody(SantaCruz,USA),therabbitanti-
humanH9252-actinantibody(SantaCruz,USA),andthehorseradishperox-
idase-conjugatedsecondaryantibodies(JacksonLaboratory,USA)were
purchased.
Cellcultureandtransfection.ThehumanhepatomacelllineHepG2
andthehumannormallivercelllineL-02wereobtainedfromtheATCC
(Manassas,VA,USA).TheHepG2-C5andHepG2-4D14celllines,in
whichtheHBVgenomewasintegrated,werekindlyprovidedbyDong-
pingXu(302HospitalofPLA).Transfectionswereperformedbyusing
Lipofectamine2000reagent(Invitrogen,USA).
RNAextractionandreal-timePCR.TotalRNAwasextractedfrom
cellsorfrozentissuesbyusingTRIzolreagent(Invitrogen,USA)accord-
ingtothemanufacturer’sinstructions.TheRNAwasreversetranscribed,
andreal-timePCRwasperformedbyusingSYBRgreenreal-timePCR
mastermix(Toyobo,Japan).Relativeexpressionwasquantifiedbyusing
thecomparativethresholdcycle(C
T
)method.
Luciferaseassay.HepG2cellswerecotransfectedwithpGLO-Smad7-
3=UTR,pGLO-Smad7-3=UTR-mut,andmiR-15afor24h.Thecelllysates
wereharvestedforadual-luciferaseassayaccordingtothemanufacturer’s
instructions(Promega,USA).Threeindependentrepeatswereper-
formed.ForanalyzingtheactivationofTGF-H9252signaling,theTGF-H9252re-
porterp3TP-lucandmiR-15awerecotransfectedintoHepG2cellsfor24
h,andthecelllysateswereharvestedforluciferaseassays.
Immunoblotting.ProteinsamplesweresubjectedtoSDS-PAGEand
blottedwiththeindicatedantibodies.Aftersampleswerewashedwith
phosphate-bufferedsaline(PBS)–Tween(PBST)threetimes,thehorse-
radishperoxidase-conjugatedsecondaryantibodywasaddedfor1h.Pro-
teinbandswerevisualizedbyusingEnlightWesternblottingreagents
(EngreenBiosystem,China).
Caspaseactivityassay.HepG2andL-02cellswereplatedintoa96-well
plateandtreatedasindicated.Thecelllysateswereharvestedforcaspase
activityassayswithCaspase-Glo3/7reagents(Promega,USA).Thelumines-
cenceofeachsamplewasmeasuredwithaplate-readingluminometer.
Flowcytometry.HepG2cellsweretransfectedwithpHBV1.3,
pHBV1.3-mut,orpcDNA3.1for24handthentreatedwithTGF-H92521(10
H9262g/ml)for48h.ThecellsweresubjectedtoannexinVandpropidium
iodide(PI)stainingforflowcytometryanalysis.
ConstructionofHepG2-4D14-shSmad7celllines.Thelentiviral
plasmidsforexpressingshorthairpinRNAs(shRNAs)targetingSmad7or
controlshRNAandthelentiviralpackagingsystemwerepurchasedfrom
Sigma-Aldrich(geneidentificationno.4092).LentivirusesforeachshRNA
wereproducedaccordingtothemanufacturer’sinstructions.HepG2-4D14
cellswereinfectedwiththelentivirusesinthepresenceofPolybrene(8H9262g/ml)
for48h.Thecellswerethenselectedwithpuromycin(1H9262g/ml)andnamed
HepG2-4D14-shSmad7orHepG2-4D14-shNC(negativecontrol).
Cellviabilityanalysis.HepG2,HepG2-4D14,andHepG2-4D14-
shSmad7cellswereseededinto96-wellplatesandtreatedwithorwithout
TGF-H9252(10H9262g/ml)fortheindicatedtimes.Thecellswerethenharvested
andsubjectedtoaviabilityassayusingCellCountingKit-8(CCK-8).
Tumorigenesis.Atotalof10
7
cells(HepG2,HepG2-4D14,orHepG2-
4D14-shSmad7cells)wereresuspendedinPBSandinjectedsubcutane-
ouslyintotheleftupperflankregionsof6-to8-week-oldBALB/cnude
mice(5foreachgroup).After30days,themiceweresacrificed,thetu-
morsweredissected,andthesizeofthetumorswasmeasuredandcalcu-
latedasfollows:tumorvolume(mm
3
)H11005(LH11003W
2
)/2,whereListhelong
axisandWistheshortaxis.Allanimalsreceivedhumanecare,andthe
studyofmicewaspermittedbytheResearchEthicsCommitteeofthe
InstituteofMicrobiology(approvalno.PZ2MCAS2013002).
Immunofluorescence.Tumortissueswerefixedwith4%paraformal-
dehydeinPBSfor8hat4°C.Afterfixation,thetissueswereembeddedin
paraffin,cutwithamicrotome(Leica)to5H9262m,andaffixedontotheslides.
Theslidesweretreatedwith3%H
2
O
2
for30minat25°Candwith0.01M
sodiumcitrate(pH6.0)forantigenretrievalandpermeabilizedwithpre-
cooledacetonefor10min.Theslideswerethenblockedwith10%goat
serumfor1hat25°Candincubatedwithrabbitanti-Smad7antibodyover-
nightat4°C.Afterbeingwashed3timesinPBST,theslideswereincubated
withAlexaFluor488goatanti-rabbitIgG(HH11001L)for1hat25°C.Thesections
werecounterstainedwith4=,6-diamidino-2-phenylindole(DAPI),andim-
ageswereacquiredwithaNikonEclipse80iinstrument.
Statisticalanalysis.Differencesbetweeneachgroupweredetermined
byusingStudent’sttest.APvalueofH110210.05wasconsideredsignificant.
ThedegreeofassociationbetweenvariableswasdeterminedbySpear-
man’snonparametriccorrelation.
RESULTS
MiR-15adownregulatesSmad7expressionbytargetingits3=
UTR.Inourpreviousstudy,wefoundthatHBVcandownregulate
thelevelofmiR-15a,whichhasbeenreportedtoactasaputative
tumorsuppressorbytargetingBcl-2,CCND1,andWNT3A(13).
Liuetal.
2740jvi.asm.orgMarch2015Volume89Number5JournalofVirology
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TofurtherunderstandthesignificanceoftheHBV/miR-15apath-
way,wetriedtoidentifyothertargetsofmiR-15a.Byanalysiswith
TargetScanatthemiRBasewebsite(http://www.mirbase.org/),
severalgenes,suchastheDEDD,CDC25A,Smad7,E2F3,E2F7,
andPLAGL1genes,werepredictedtobethetargetsofmiR-15a.
Toconfirmtheabove-mentionedpredictions,wetransfected
HepG2cellswithmiR-15aandexaminedtheexpressionofthese
geneswithCCND1asapositivecontrol.AsshowninFig.1A,the
mRNAlevelsofCDC25A,Smad7,andE2F7weresignificantly
decreasedinHepG2cellstransfectedwithmiR-15a.Wechose
Smad7forfurtherstudysinceitisanimportantregulatorof
TGF-H9252signaling.First,weexaminedtheproteinlevelofSmad7in
miR-15a-transfectedcells.Theimmunoblottingdatashowedthat
theproteinlevelofSmad7wassignificantlyreducedinmiR-15a-
transfectedcells(Fig.1B).WethenlookedforthepotentialmiR-
15a-targetingsiteonSmad7byusingTargetScan.Thereisaputa-
tivemiR-15a-complementaryregionlocatedintheSmad7mRNA
3=UTR(nt1608to1627)(Fig.1C).Therefore,wegenerateda
Smad73=-UTRluciferasereporter(pGLO-Smad7-3=UTR)andits
mutant(pGLO-Smad7-3=UTR-mut),inwhichmutationsinthe
miR-15a-bindingsiteweremadeasindicatedinthelegendofFig.
1D.WetransfectedthemwithorwithoutmiR-15aintoHepG2
cells.Thecelllysateswereharvestedfordual-luciferaseassays.The
datashowedthatmiR-15acanreducetherelativeluciferaseactiv-
ityofpGLO-Smad7-3=UTRbutnotthatofpGLO-Smad7-3=UTR-
mut(Fig.1E).TheseresultssuggestedthatSmad7isanoveltarget
ofmiR-15athatcandownregulatetheexpressionofSmad7by
targetingits3=UTR.
HBVupregulatesSmad7expressionbydownregulating
miR-15ainhepatocytes.Inapreviousstudy,wefoundthatHBV
causesareductioninthelevelofmiR-15a.Wewonderwhether
HBVcouldupregulateSmad7expressionbydownregulating
miR-15a.First,weconfirmedthattheexpressionlevelofmiR-15a
inHBVtransgenicHepG2-C5andHepG2-4D14cellswasmuch
lowerthanthatinHepG2cells(Fig.2A).Wethenanalyzedthe
levelsofSmad6andSmad7mRNAsandtheamountsofSmad6
andSmad7proteinsinHepG2,HepG2-C5,andHepG2-4D14
cells.ThedatashowedthatbothSmad7mRNA(Fig.2B,left)and
Smad7protein(Fig.2B,right)levelsinHepG2-C5andHepG2-
4D14cellsweresignificantlyhigherthanthoseinHepG2cells,
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FIG1MiR-15adownregulatesSmad7expressionbytargetingits3=UTR.(A)HepG2cellsweretransfectedwithmiR-15aorarandomizedoligonucleotideas
acontrolfor48h.TotalRNAwasextractedandquantifiedbyreal-timePCRfortheexpressionofpredictedtargetgenes.(B)HepG2cellsweretransfectedwith
miR-15aorarandomizedoligonucleotideasacontrolfor48h.Celllysateswereharvestedforimmunoblottingwithrabbitanti-humanSmad7antibody.(C)The
predictedmiR-15a-bindingsequencesarelocatedinthe3=UTRofSmad7mRNA.Thematchednucleotidesareindicatedwithaline.(D)Mutationsonthe3=
UTRofSmad7weremadeinthepredictedregionofmiR-15a-bindingsites(namedSmad7-3=UTR-mut).Themutatednucleotidesareshowninboldfaceitalic
type.(E)HepG2cellswerecotransfectedwiththemiR-15amimicandpGLO-Smad7-3=UTRorpGLO-Smad7-3=UTR-mut.Thecelllysateswereharvestedfor
dual-luciferaseassays.Therelativeluciferaseactivitywasquantified.H11569H11569,PH110210.01.
HBVInhibitsApoptosisthroughtheTGF-H9252Pathway
March2015Volume89Number5jvi.asm.org2741JournalofVirology
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whiletherewasnoobviousdifferenceinSmad6expressionlevels
intheabove-describedcells.Furthermore,wetransfected
pHBV1.3orpHBV1.3-mutintoHepG2andL-02cellsandana-
lyzedtheamountsofSmad7mRNAandprotein.Thedatashowed
thattheamountsofbothSmad7mRNA(Fig.2C)andprotein
(Fig.2D)wereupregulatedinpHBV1.3-transfectedcellscom-
paredtothoseincontrolcellsandpHBV1.3-mut-transfectedcells.
TheseresultssuggestedthatHBVcanupregulatetheexpressionof
Smad7.
HBVupregulatesSmad7expressionbydownregulating
miR-15ainvivo.TofurtherconfirmthatHBVcanupregulatethe
expressionofSmad7invivo,weexaminedtheamountofSmad7
mRNAinHBVtransgenicBALB/cmicebyreal-timePCR.The
datashowedthattherelativeamountofSmad7mRNAinliver
tissueofHBVtransgenicBALB/cmicewas2-foldhigherthanthat
incontrolmice(PH110210.01)(Fig.3A).Wenextexaminedthecor-
relationbetweenmiR-15aandSmad7mRNAinlivertissuesam-
plesfromHCCpatientswhowereallchronicHBVcarriers.As
showninFig.3B,therewasanegativecorrelationbetweenmiR-
15aandSmad7mRNAlevels.Wethenanalyzedthelevelsof
Smad7mRNAandHBVtranscriptsinlivertissuesamplesfrom
HCCpatientsbyreal-timePCR.Thedataindicatedthattherewas
apositivecorrelationbetweenthelevelsofHBVmRNAsandthe
levelofSmad7mRNAinlivertissuesamplesofHCCpatients(Fig.
3C).Inaddition,wecollectedthelivertumorandadjacentnon-
tumorouslivertissues(ANLTs)from5patientsandexamined
Smad7proteinlevels.ThedatashowedthatthelevelofSmad7
proteinintumorswashigherthanthatinadjacentnontumorous
livertissuesofeachpatient(Fig.3D,top).Moreimportantly,the
levelofSmad7proteinwaspositivelycorrelatedwiththeamount
ofHBVmRNAinpatients(Fig.3D).
MiR-15aenhancesTGF-H9252signalingbydownregulating
Smad7.ItisknownthatthatSmad7actsasanantagonistof
TGF-H9252signaling(16).Therefore,weexaminedwhethermiR-15a
couldenhanceTGF-H9252signalingbydownregulatingSmad7.
HepG2cellsweretransfectedwithanmiR-15amimic,anmiR-15a
inhibitor,orarandomizedoligonucleotideasacontrolandthe
TGF-H9252luciferasereporterp3TP-luc,followedbytreatmentwith
TGF-H92521.Thecelllysateswereharvestedforluciferaseassays.As
showninFig.4A,themiR-15amimicenhancedtheactivationof
theTGF-H9252luciferasereporter,whilethemiR-15ainhibitorinhib-
itedtheactivationofTGF-H9252signaling.Wethentransfected
HepG2cellswithpFLAG-CMV2-Smad7andtreatedthecellswith
TGF-H92521.WeanalyzedtheamountofSmad2intotalcellextracts
andinthenucleusbyimmunoblotting.Thedatashowedthatthe
amountofSmad2inthenucleusinSmad7-overexpressingcells
waslowerthanthatincontrolcells,whilethetotalamountsof
Smad2weresimilarinbothgroups(Fig.4B).Wecheckedthe
amountofSmad2inmiR-15amimic-transfectedcells.Asshown
inFig.4C,miR-15aenhancedthetranslocationofSmad2intothe
nucleusincellstreatedwithTGF-H92521.However,theoverexpres-
sionofSmad7counteractedtheeffectofmiR-15aonthenuclear
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0.0
0.5
1.0
1.5
2.0
Smad6
Smad7
R
e
la
t
iv
e
m
R
N
A
le
v
e
ls
Smad7
β-actin
Smad6
1.01.632.05
1.00.941.13
FIG2HBVupregulatesSmad7expressionbydownregulatingmiR-15ainhepatocytes.(AandB)TotalmRNAsofHepG2,HepG2-C5,andHepG2-4D14cells
wereextractedandsubjectedtoreal-timePCRformiR-15a(A)andSmad6andSmad7mRNAs(B,left),andthecelllysatesfromtheabove-describedcelllines
wereharvestedforimmunoblottingwithSmad6andSmad7antibodies(B,right).(CandD)HepG2andL-02cellsweretransfectedwithpHBV1.3,pHBV1.3-
mut,orpcDNA3.1asthecontrolfor48h.TheamountofSmad7mRNAwasquantifiedbyreal-timePCR(C),andtheproteinlevelofSmad7wasdetectedby
immunoblotting(D).H11569H11569,PH110210.01.
Liuetal.
2742jvi.asm.orgMarch2015Volume89Number5JournalofVirology
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translocationofSmad2(Fig.4D).ThesedataindicatedthatmiR-
15aenhancedTGF-H9252signalingandpromotedSmad2transloca-
tionintothenucleusthroughdownregulatingtheexpressionof
Smad7.
HBVinhibitsTGF-H9252signalingbydownregulatingmiR-15a.
Basedontheabove-describedresults,whichdemonstratedthat
HBVcouldupregulatetheexpressionofSmad7,wesoughtto
determinewhetherHBVcouldinhibitTGF-H9252signaling.HepG2
cellsweretransfectedwithpHBV1.3orpHBV1.3-muttogether
withtheTGF-H9252reporterp3TP-lucandthentreatedwithTGF-H92521.
Thecellswereharvestedforluciferaseassaysandimmunoblot-
ting.AsshowninFig.5A,therelativeluciferaseactivityin
pHBV1.3-transfectedcellswassignificantlyreducedcompared
withthatincontrolorpHBV1.3-mut-transfectedcellstreated
withTGF-H92521.Consistently,theamountofSmad2inthenucleus
wasgreatlyreducedinpHBV1.3-transfectedcellscomparedto
thatincontrolorpHBV1.3-mut-transfectedcells(Fig.5B).To
furtherdeterminewhetherHBVupregulatesthelevelofSmad7by
downregulatingmiR-15a,wetransfectedHepG2cellswith
pHBV1.3withorwithoutthemiR-15amimic,followedbytreat-
mentwithTGF-H92521.Thecelllysateswereharvestedforimmuno-
blottingwiththeindicatedantibodies.AsshowninFig.5C,the
levelofSmad7wasincreasedinpHBV1.3-transfectedcellsbut
returnedtotheoriginallevelinpHBV1.3-andmiR-15a-cotrans-
fectedcells,suggestingthatmiR-15acancounteracttheeffectof
pHBV1.3ontheupregulationofSmad7.Consistently,asshownin
Fig.5D,theamountofSmad2inthenucleuswassignificantly
lowerinpHBV1.3-transfectedcellthanthatincontrolcells,butin
AB
C
D
BA
L
B/
c
H
B
V
t
r
a
n
s
g
eni
c
B
A
LB
/
c
0.0
0.5
1.0
1.5
2.0
2.5
R
e
l
at
i
v
eam
ount
of
S
m
ad7m
R
N
A
Case1Case2Case3Case4Case5
AN
L
T
AN
L
T
AN
L
T
AN
L
T
AN
L
T
TTTTT
Smad7
β-actin
1.01.5
Patient
1.01.41.03.11.02.41.03.3
Case1Case2Case3Case4Case5
0.0
0.5
1.0
1.5
2.0
ANLTT
R
e
l
a
ti
v
e
a
m
o
u
n
to
fH
B
V
tr
a
n
s
c
r
i
p
ts
R2=0.690
n=40
0
10
20
30
40
50
60
70
80
050100150200
R
el
a
t
i
v
eam
ou
n
t
of
S
m
ad7
m
R
N
A
RelativeamountofmiR-15a
R2=0.662
n=40
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0102030405060
Re
la
t
i
v
e
a
m
o
u
n
t
o
f
S
m
a
d
7
m
RNA
RelativeHBVtranscripts
FIG3HBVupregulatesSmad7expressionbydownregulatingmiR-15ainvivo.(A)TotalRNAsextractedfromlivertissuesofHBVtransgenicBALB/cmice
(nH110053)orcontrolBALB/cmice(nH110053)weresubjectedtoreal-timePCRtodetecttheamountofSmad7mRNA.(B)TotalRNAsfromliverspecimensofHCC
patientswereextractedandsubjectedtoreal-timePCRformiR-15aandSmad7mRNA.ThecorrelationbetweenmiR-15aandSmad7mRNAinsamplesfrom
HCCpatientswasanalyzedbyaSpearmanranktest.Correlationcoefficient(R)valueswerecalculated.(C)TotalRNAsfromliverspecimensofHCCpatients
wereextractedandsubjectedtoreal-timePCRforSmad7mRNAandHBVtranscripts.ThecorrelationbetweenHBVtranscriptsandSmad7mRNAinHCC
patientsampleswasanalyzedbyaSpearmanranktest.Rvaluesarealsoshown.(D)ProteinlevelsofSmad7weredetectedbyimmunoblotting(top),andthe
relativeamountofHBVmRNAwasdetectedbyreal-timePCR(bottom)intumortissues(T)andadjacentnontumorouslivertissues(ANLTs).H11569H11569,PH110210.01.
HBVInhibitsApoptosisthroughtheTGF-H9252Pathway
March2015Volume89Number5jvi.asm.org2743JournalofVirology
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pHBV1.3-andmiR-15a-cotransfectedcells,itwentbacktothe
samelevelasthatincontrolcells.Wealsocheckedtheamountof
phosphorylatedSmad2inpHBV1.3-andpCMV-Myc-Smad2-
transfectedHepG2cellstreatedwithTGF-H92521.Thedatashowed
thatthelevelofphosphorylatedSmad2inpHBV1.3-andpCMV-
Myc-Smad2-cotransfectedcellswasmuchlowerthanthatin
pCMV-Myc-Smad2-transfectedcells(Fig.5E),whichsuggested
thatHBVcoulddownregulatethelevelofphosphorylatedSmad2
inHepG2cells.
HBVmakesHepG2cellsresistanttoTGF-H92521-inducedapop-
tosis.AsHBVcandownregulateTGF-H9252signalingthroughthe
miR-15a/Smad7pathway,wewonderedwhethertherewasany
correlationbetweenHBVandTGF-H9252-inducedapoptosis.First,
wetestedwhetherTGF-H9252couldinduceapoptosisinHepG2cells
bycheckingthecleavageofPARP-1.AsshowninFig.6A,PARP-1
cleavageoccurredobviouslyinTGF-H92521-treatedcells.Wethen
comparedPARP-1cleavageinHepG2,HepG2-C5,andHepG2-
4D14cellstreatedwithTGF-H92521.ThedataindicatedthatPARP-1
cleavageinHepG2-C5andHepG2-4D14cellswasmuchweaker
thanthatinHepG2cells,whichmeansthatHepG2-C5and
HepG2-4D14cellsareresistanttoapoptosisinducedbyTGF-H92521
(Fig.6B).Furthermore,wetransfectedHepG2cellswithpHBV1.3
andpHBV1.3-mutandtreatedthecellswithTGF-H92521.H9001sshownin
Fig.6C,PARP-1cleavagewasreducedinpHBV1.3-transfected
cellscomparedtothatincontrolcells,whiletherewasnoobvious
reductioninpHBV1.3-mut-transfectedcells.Next,wetransfected
HepG2cellswithpHBV1.3withorwithoutmiR-15aandtreated
thecellswithTGF-H92521.ThedatashowedthatPARP-1cleavagewas
reducedinpHBV1.3-transfectedcellscomparedtothatincontrol
cells,whileinpHBV1.3-andmiR-15a-cotransfectedcells,PARP-1
cleavagewasrecoveredtoacertainlevel(Fig.6D).Wealsoexamined
caspase3/7activityinHepG2andL-02cells,whichweretransfected
withpHBV1.3,pHBV1.3-mut,orthecontrolplasmidandtreated
withTGF-H92521.Thedatashowedthatthelevelofcaspase3/7activity
inpHBV1.3-transfectedcellswaslowerthanthoseincontrolcells
andpHBV1.3-mut-transfectedcells(Fig.6E).Finally,weper-
formedflowcytometryanalysiswithannexinVandPIstainingto
monitorapoptoticcells.AsshowninFig.6F,afterTGF-H92521treat-
ment,thepercentageofapoptoticcellsinpHBV1.3-transfected
cellswas3-foldlowerthanthatincontrolcells,whilethepercent-
ageofapoptoticcellsinpHBV1.3-mut-transfectedcellswasalso
reducedbutnotasdramaticallyasinpHBV1.3-transfectedcells.
TheseresultssuggestedthatHBVcouldinhibitTGF-H9252-induced
apoptosisthroughthemiR-15a/Smad7pathway.
HBVpromotestumorigenesisthroughinhibitingTGF-H9252
signalingbyupregulatingSmad7.Ithasbeenreportedthat
TGF-H92521signalingisinvolvedincellproliferationandtumorigen-
esis(21).WewonderedwhetherHBVcouldpromotetumorigen-
AB
CD
0
2
4
6
8
10
Control
miR-15amimic
miR-15ainhibitor
R
e
l
a
ti
v
e
L
u
c
i
fe
r
a
s
e
a
c
ti
v
i
ty
TGF-β1
Smad2
LaminB
TotalNucleus
miR-15a
FLAG-Smad7
1.01.891.08
FLAG-Smad7
Smad2
LaminB
TotalNucleus
FLAG-Smad7
FLAG-Smad7
1.00.216
Smad2
β-actin
LaminB
miR-15a
control
TotalNucleus
1.01.88
FIG4MiR-15aenhancesTGF-H9252signalingbydownregulatingSmad7.(A)HepG2cellsweretransfectedwithanmiR-15amimic,anmiR-15ainhibitor,ora
randomizedoligonucleotideasacontrol,togetherwiththeTGF-H9252luciferasereporterp3TP-lucandrenillaluciferaseplasmidpRL-TK.Thecelllysateswere
harvestedforfireflyluciferaseandrenillaluciferaseactivityassays.Therelativeluciferaseactivitywasquantified.(B)HepG2cellsweretransfectedwithpcDNA3.1
orpCMV-FLAG-Smad7for24handthentreatedwithTGF-H92521for24h.Thetotalcelllysateandthenuclearfractionweresubjectedtoimmunoblottingwiththe
indicatedantibodies.(C)HepG2cellsweretransfectedwiththemiR-15amimicorarandomizedoligonucleotideasacontrolfor24handthentreatedwith
TGF-H92521for24h.Immunoblottingwasperformedasdescribedabove.(D)HepG2cellsweretransfectedwiththemiR-15amimicwithorwithout
FLAG-Smad7for24handthentreatedwithTGF-H92521for24h.Immunoblottingwasperformedasindicated.TherelativeamountofSmad2inthenucleus
wasquantified.H11569,PH110210.05.
Liuetal.
2744jvi.asm.orgMarch2015Volume89Number5JournalofVirology
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esisthroughinhibitingTGF-H9252signaling.Toaddressthisquestion,
wefirstmeasuredthecellviabilitiesofHepG2andHepG2-4D14
cellstreatedwithTGF-H92521fortheindicatedtimes.AsshowninFig.
7A,theviabilityofHepG2-4D14cellsisbetterthanthatofHepG2
cellsafterTGF-H92521treatment.WetheninjectedHepG2,HepG2-
C5,andHepG2-4D14cellssubcutaneouslyintonudemiceand
measuredthesizeoftumorsatdifferenttimepoints.Thedata
showedthatHepG2-C5andHepG2-4D14cellsformedlargertu-
morsinnudemicethandidHepG2cells(Fig.7B).Wedissected
thetumorsfromeachgroupandmeasuredthelevelsofmiR-15a
andSmad7.AsshowninFig.7C,thetumorsgeneratedfrom
HepG2-4D14cellsexpressedlowerlevelsofmiR-15a(Fig.7C,left)
andhigherlevelsofSmad7mRNAandprotein(middleandright)
thandidthetumorsgeneratedfromHepG2cells.Inordertocon-
firmthefunctionofSmad7intumorigenesisofHBV-positive
cells,wegeneratedtheSmad7knockdowncelllinesshSmad7#1
andshSmad7#2,whichwerederivedfromHepG2-4D14cells.The
efficiencyofSmad7knockdownintheabove-describedcelllines
wasdeterminedbyimmunoblotting(Fig.7D,bottom).Thedata
fromthecellviabilityassaysshowedthattheviabilityinSmad7
knockdowncellswasreducedcomparedtothatincontrolcells
treatedwithTGF-H9252(Fig.7D,top).Theresultsfromtumorforma-
tionassayswithnudemiceshowedthatthetumorsgenerated
fromshSmad7#1andshSmad7#2cellswereobviouslysmaller
thanthosegeneratedfromcontrolcells(Fig.7E),whichsuggested
thatSmad7facilitatedthetumorigenesisofHepG2-4D14cells.In
conclusion,werevealedthatHBVcaninhibitapoptosisandpro-
motetumorigenesisthroughinhibitingTGF-H9252signalingbyup-
regulatingSmad7.
DISCUSSION
HCCisamongthemostfrequentformsofcancer,anditsinci-
denceisincreasingworldwidebecauseofhepatitisBandCvirus
infection(22).HBVinfectionaccountsformorethan60%of
HCCpatientsindevelopingcountriesandasmuchas80%ofall
HCCcasesinChina(23,24).However,therearestillsomeunad-
dressedquestionsregardingthemechanismofHBVinfection
causingHCC.Inthisstudy,weproposedthatHBVmRNAsactas
“ceRNAs”(competingendogenousRNAs)formiR-15atoregu-
ABC
DE
TGF-β1
0
2
4
6
8
pHBV1.3-mut
pcDNA3.1
pHBV1.3
R
el
at
i
v
e
Luc
i
f
er
as
eac
t
i
v
i
t
y
Smad2
LaminB
TotalNucleus
pHBV1.3
pHBV1.3-mut
1.00.491.02
Smad7
pHBV1.3
miR-15a
β-actin
1.02.221.41
Smad2
LaminB
TotalNucleus
pHBV1.3
miR-15a
1.00.411.09
p-Myc-Smad2
LaminB
TotalNucleus
pHBV1.3
Myc-Smad2
β?actin
1.00.38
Myc-Smad2
FIG5HBVinhibitsTGF-H9252signalingbydownregulatingmiR-15a.(A)HepG2cellsweretransfectedwithpHBV1.3,pHBV1.3-mut,orpcDNA3.1asthecontrol,
togetherwiththeTGF-H9252luciferasereporterp3TP-lucandrenillaluciferaseplasmidpRL-TK.Thecelllysateswereharvestedforfireflyluciferaseandrenilla
luciferaseactivityassays.Therelativeluciferaseactivitywasquantified.(B)HepG2cellsweretransfectedwithpHBV1.3orpcDNA3.1asthecontrol,withor
withoutthemiR-15amimic,for24handthentreatedwithTGF-H92521for24h.Thetotalcelllysateandnuclearfractionweresubjectedtoimmunoblottingwiththe
indicatedantibodies.(C)HepG2cellsweretransfectedwithpHBV1.3orpHBV1.3-mutfor24handthentreatedwithTGF-H92521for24h.Immunoblottingwas
performedasindicated.(D)HepG2cellsweretransfectedwithpHBV1.3withorwithoutthemiR-15amimicfor24handthentreatedwithTGF-H92521for24h.The
celllysatesweresubjectedtoimmunoblotting.(E)HepG2cellsweretransfectedwithpHBV1.3andMyc-Smad2for24handthentreatedwithTGF-H92521for24h.
Immunoblottingwasperformedasdescribedabove.TherelativeamountofSmad2inthenucleuswasquantified.H11569H11569,PH110210.01.
HBVInhibitsApoptosisthroughtheTGF-H9252Pathway
March2015Volume89Number5jvi.asm.org2745JournalofVirology
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lateTGF-H9252signaling,whichcontributestothedevelopmentof
HBV-relatedHCC.
Recently,ceRNAshavebeencharacterizedbytheirabilityto
competeformiRNAbindingandalleviatetherepressiveeffectof
miRNAsontheirmRNAtargets(25).MoreandmoreceRNAs
havebeenidentifiedandfoundtoparticipateinmiRNA-depen-
dentcrosstalkandproducerobustnetworks(26–30).Deregula-
tionofceRNAsmayleadtocancer(31).Forexample,Hmga2can
AB
D
E
C
CleavedPARP-1
β-actin
TGF-β
116
89
Kd
F
0.0
0.5
1.0
1.5
2.0
2.5
3.0
pcDNA3.1
pHBV1.3
pHBV1.3-mut
R
e
l
at
i
v
eC
as
p
as
e3/
7ac
t
i
v
i
t
y
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
pcDNA3.1
pHBV1.3
pHBV1.3-mut
R
e
l
a
t
i
v
e
C
a
s
pas
e3/
7ac
t
i
v
i
t
y
L-02cells
TGF-βTGF-β
HepG2cells
CleavedPARP-1
β-actin
116
89
TGF-β
Kd
CleavedPARP-1
β-actin
TGF-β
116
89
Kd
pHBV1.3
pHBV1.3-mut
CleavedPARP-1
β-actin
TGF-β
116
89
Kd
pHBV1.3
miR-15a
PI
Annexin
-TGF-β1+TGF-β1
pcDNA3.1
pHBV1.3
pHBV1.3-mut
1.13%1.25%
3.00%0.754%
0.284%
2.51%4.03%
1.49%
1.96%8.10%
3.00%13.8%
pcDNA3.1pHBV1.3pHBV1.3-mut
0
2
4
6
8
10
12
14
16
-TGF-β1+TGF-β1
P
e
r
c
ent
a
geof
apopt
ot
i
c
c
e
l
l
s
FIG6HBVmakesHepG2cellsresistanttoTGF-H9252-inducedapoptosis.(A)HepG2cellsweretreatedwithorwithoutTGF-H92521for48h.Thecelllysateswere
harvestedforimmunoblottingwithPARP-1antibodyorH9252-actinasaninternalcontrol.(B)HepG2,HepG2-C5,andHepG2-4D14cellsweretreatedwithor
withoutTGF-H92521for48h.Thecelllysateswereharvestedforimmunoblottingwiththeindicatedantibodies.(C)HepG2cellsweretransfectedwithpHBV1.3or
pHBV1.3-mutfor24handthentreatedwithTGF-H92521for24h.Thecelllysateswereharvestedforimmunoblottingwiththeindicatedantibodies.(D)HepG2cells
weretransfectedwithpHBV1.3orpHBV1.3-mutfor24handthentreatedwithTGF-H92521for24h.Thecelllysateswereharvestedforimmunoblottingwiththe
indicatedantibodies.(E)HepG2(left)andL-02(right)cellsweretransfectedwithpHBV1.3orpcDNA3.1for24handthentreatedwithTGF-H92521for24h.The
celllysateswereharvestedandsubjectedtoacaspase3/7activityassay.(F)HepG2cellsweretransfectedwithpHBV1.3,pHBV1.3-mut,orpcDNA3.1asacontrol
for24handthentreatedwithTGF-H92521for48h.(Left)ThecellsweresubjectedtoannexinVandPIstainingforflowcytometryanalysis.(Right)Thepercentage
ofapoptoticcellwascalculated.H11569,PH110210.05;H11569H11569,PH110210.01.
Liuetal.
2746jvi.asm.orgMarch2015Volume89Number5JournalofVirology
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AB
C
D
0122448
80
85
90
95
100
HepG2
HepG2-4D14
TimeBè:?Bé
C
e
llv
i
a
b
ilit
y
(
%
)
TGF-β
0244872
70
75
80
85
90
95
100
sh-NC
shSmad7#1
shSmad7#2
TimeBè:?Bé
C
e
llv
ia
b
ilit
y
(
%
)
TGF-β
H
epG
2
H
epG
2-
4D
14
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
R
e
l
a
t
i
v
ehs
a-
m
i
R
-
15al
e
v
e
l
s
H
epG
2
H
epG
2-
4D
14
0
1
2
3
4
5
6
7
8
9
R
e
l
a
ti
v
e
m
R
N
A
l
e
v
e
l
s
o
f
S
M
A
D
7
E
10μm10μm
HepG2HepG2-4D14
Negative
Smad7
Tumor
10μm10μm
β-actin
Smad7
1.00.470.38
7142128
0
100
200
300
400
500
sh-NC
shSmad7#1
shSmad7#2
Days
Tu
m
o
r
s
i
z
e
(
m
m
3
)
sh-NC
shSmad7#1
shSmad7#2
7142128
0
100
200
300
400
500
600
HepG2
HepG2-C5
HepG2-4D14
Days
T
u
mo
r
v
o
l
u
m(
mm
3
)
HepG2
HepG2-4D14
HepG2-C5
FIG7HBVpromotestumorigenesisthroughinhibitingTGF-H9252signalingbyupregulatingSmad7.(A)HepG2andHepG2-4D14cellsweretreatedwithTGF-H92521
for12h,24h,and48h.CellviabilitywasdetectedbyaCCK-8assay.(BandC)HepG2,HepG2-C5,andHepG2-4D14cellswereinjectedsubcutaneouslyinto
BALB/cnudemice(5mice/group).(B,left)Thesizeoftumorswasmeasuredattheindicatedtimesafterinjection.(Right)Atday30,thetumorsweredissected
andphotographed.(C,left)TheRNAsoftumorsgeneratedfromHepG2andHepG2-4D14cellsweresubjectedtoreal-timePCRformiR-15aandSmad7mRNA.
(Right)ImmunofluorescenceanalysisoftumortissueswasperformedwithSmad7antibodyorrabbitIgGasanegativecontrol.(D)Smad7knockdown
HepG2-4D14cells(HepG2-4D14shSmad7#1[shSmad7#1inshort]andHepG2-4D14shSmad7#2[shSmad7#2inshort])orcontrol(HepG2-4D14sh-NC
[sh-NCinshort])cellsweregeneratedasdescribedinMaterialsandMethods.ThecellsweretreatedwithTGF-H92521fortheindicatedtimes.(Top)Cellviabilitywas
detectedbyaCCK-8assay.(Bottom)TheproteinlevelofSmad7incellswascheckedbyimmunoblotting.(E)HepG2-4D14Sh-NC,HepG2-4D14shSmad7#1,
orHepG2-4D14shSmad7#2cellswereinjectedsubcutaneouslyintoBALB/cnudemice.(Top)Thesizeoftumorswasmeasuredattheindicatedtimesafter
injection.TheresultsarepresentedasmeansH11006standarddeviationsfrom5miceforeachgroup.(Bottom)Thetumorsweredissectedatday30andphotographed.
March2015Volume89Number5jvi.asm.org2747JournalofVirology
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operateasaceRNAforthemiRNAlet-7topromotelungcancer
progression(32).BasedonthemechanismofceRNAscompeting
formiRNAbinding,weproposedthatHBVmRNAsmayactas
“ceRNAs”ofmiR-15atoregulateTGF-H9252signaling.Ithasbeen
suggestedHBVmRNAsactingasceRNAsmayrepresenttheviral
strategyofHBVtoexploititscompactgenomeforthedevelop-
mentofHBV-relatedHCC.Inapreviousstudy,weshowedthat
HBVmRNAs(nt1368to1383)canfunctionasmiR-15asponges
andincreasetheexpressionlevelofBcl-2,whichisoneofthe
miR-15atargets.Here,weidentifiedthatSmad7isanoveltargetof
miR-15a.HBVmRNAsreducedthemiR-15aexpressionleveland
increasedthelevelofSmad7todownregulateTGF-H9252signaling.
TGF-H9252isknowntobeamultifunctionalfactorthatplaysacritical
roleinvariousaspectsofliverpathogenesis,includingchronic
HBV/hepatitisCvirus(HCV)infection,cirrhosis,andtumori-
genesis(33,34).TGF-H9252signalingcouldhavecytostaticeffectsdur-
ingliverdamageaswellascarcinogenesis.Inculturedhepatocytes,
TGF-H9252cantriggerapoptosisandanepithelial-mesenchymaltran-
sition(EMT)viatheinductionofreactiveoxygenspecies(ROS)
andtheproapoptoticproteinBIM(15,35).WefoundthatHBV
caninhibitTGF-H9252signalingthroughupregulatingSmad7.This
mayexplainwhytheHBVtransgeniccelllineHepG2-4D14isless
sensitivetoTGF-H9252-inducedapoptosis.
HBVhasbeenfoundtodownregulateothermiRNAstocon-
trolvarioussignalingpathways.Forexample,HBVmRNAcan
reducethelevelofmiR-122bydirectabsorptiontopromote
hepatocellularcarcinomatumorgrowth(11),andtheHBxpro-
teinmodulatesthetumorsuppressormiR-520b,accelerateshepa-
tocarcinogenesis,andrepressesmiR-148atoinhibittheAKT/
ERK/FOXO4/ATF5pathway(9,36).Ithasbeenreportedthat
miR-15aplaysimportantrolesinapoptosis,cellproliferation,and
tumorigenesisbytargetingdifferentmRNAsinvariouscancers
(13,37,38).Inthisstudy,weidentifiedthatHBVcaninhibit
TGF-H9252signalingbydownregulatingmiR-15aandupregulating
Smad7.Itisworthfurtherinvestigatingwhetherthereisanycross
talkbetweentheHBV/miR15a/Smad7-regulatedTGF-H9252pathway
andotherpathways,suchastheHBV/miR-122/PBForHBx/p53
signalingpathway,topromotecellproliferationandtumorigene-
sis.Inaddition,weconfirmedthatlevelsofothermiRNAs,suchas
miR-422a,miR-210,andmiR-7-5p,werealsoreduced,asre-
portedpreviouslyforHBVtransgeniccells(39).However,the
correlationofHBVinfectionwiththeexpressionofthesemiRNAs
aswellasitsbiologicalsignificanceneedtobefurtherstudied.
Inconclusion,wedemonstratethatHBVmRNAsactas
ceRNAsofmiR-15aandregulateTGF-H9252signalinginthedevelop-
mentofHBV-relatedHCC.Ourstudyprovidesevidencetosup-
porttheideathatviralRNAscanexerttheirfunctionsasregula-
toryfactorstowardmiRNA.Weproposethattheremaybemore
viralRNAsservingasceRNAsinvolvedinvariousaspectsofcel-
lularprocesses.However,thisneedsfurtherinvestigation.
ACKNOWLEDGMENTS
ThisworkwassupportedbytheMinistryofScienceandTechnologyofChina
(2012CB519003,2013ZX10004611,2011CB504705,2015CB910502,and
2012ZX10004501-001-003)andtheNationalNaturalScienceFoundationof
China(81272272).
XinYeisaprincipalinvestigatoroftheInnovativeResearchGroupof
theNationalNaturalScienceFoundationofChina(81321063).
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