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2008 Suppression of integrin atb6 by RNA interference in colon cancer cells inhibits

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Suppressionofintegrinatb6byRNAinterferenceincoloncancercellsinhibits

extracellularmatrixdegradationthroughtheMAPKpathway

JiayongWang1,ZhaoyangZhang1,KesenXu1,XiaohuiSun1,GuangyunYang1,WeiboNiu1,EnyuLiu1,

ChengPeng1,PengfeiLin1,JianWang1,RongChen2,MichaelAgrez3andJunNiu1*

1DepartmentofGeneralSurgery,QiLuHospital,ShandongUniversity,Jinan,Shandong,China

2InstituteofMedicalPathophysiology,ShandongUniversity,Jinan,Shandong,China

3NewcastleBowelCancerResearchCollaborative,HunterMedicalResearchInstitute,JohnHunterHospitaland

FacultyofMedicineandHealthSciences,TheUniversityofNewcastle,Callaghan,NewSouthWales,Australia

Integrinatb6playsaveryimportantroleintheprogressionofco-

loncancercellsandisnowdefinedasanovel,independentprog-

nosticindicatorforaggressivecoloncancerinhumans.Herein,we

usetheRNAinterferingtechnologytodownregulatetheexpres-

sionofatb6incoloncancercells.Ourdatademonstratethatplas-

midvectorbasedshRNAcaneffectivelydown-regulateatb6

expressioninproteinandmRNAlevels.Supressionofintegrin

atb6inhibitsthephosphorylationandnonphosphorylationlevelof

ERK1/2,thesecretionofuPA,pro-MMP-9andpro-MMP-2intu-

morconditionedmedium,andmoreimportant,inhibitsMAPK-

dependent[3H]labeledcollagenIVdegradationviatheplasmino-

genactivationcascade.Ourstudydemonstratesinvitrothat

supressionofintegrinatb6inhibitsextracellularmatrixdegrada-

tionthroughtheMAPKpathway.

'2008Wiley-Liss,Inc.

Keywords:coloncancer;integrinatb6;MAPkinase;RNA

interference

Cancercellinvasionandmetastasisareregardedasmultistep

phenomena,involvingtheproteolyticdegradationoftheextracel-

lularmatrix(ECM),alteredcelladhesionandthephysicalmove-

mentofcancercells.Suchmigratoryandtissueremodelingevents

involvecomplexinteractionsbetweenintegrinsandmatrix

degradingproteasesystems,suchastheplasminogenactivation

system.1TheintegrinsprovidedirectlinkswiththeECM,func-

tioningasbidirectionaltransducersofextra-andintracellularsig-

nals,andthuspotentiallymodulatingcancercellinvasion.2

Integrinatb6isrestrictedlydistributedtoepithelialcells.Itis

primarilyexpressedduringfetaldevelopmentandwoundhealing.

Upregulationoftheintegrinatb6isfoundinsomeepithelialcar-

cinomas,mostnotablyinorally-derivedsquamouscellcarcino-

mas,3–5wherethereceptortypicallydisplaysafocallylocalized

distributionattheinfiltratingedgeoftumorislands.Recently,the

mostcompellingfindingisthatatb6isdefinedasanovel,inde-

pendentprognosticindicatorforaggressivecoloncancerandgas-

triccarcinomainhumans.6,7Theintegrinatb6mayservetoiden-

tifypatientsthatareathigherriskofdevelopingmetastaticdisease

andemergesasanattractivenewcandidateasatherapeutictarget

formetastaticcoloncarcinoma.

ManyproteinasesarecapableofdegradingECMcomponents,

buttheproteinasesystemprimarilyresponsibleforECMdegrada-

tioninvivoarematrixmetalloproteinases(MMPs)andplasmino-

genactivator(PA)systems.8,9MMP-2andMMP-9havebeen

implicatedtoplayaroleincolorectalcancerprogression,invasion

andmetastasisinanimalmodelsandpatients.10Urokinaseplas-

minogenactivator(uPA)isfoundincellularstructuresatthelead-

ingedgeofmigratingcellsthatareinvolvedinadhesion,migra-

tion,invasionandintravasationandisconsideredtobeamarker

formalignancyincoloncancer.11,12

Inourpreviouswork,wehavereportedtherelationship

betweenintegrinatb6andMMP-9incoloncancer.Wereported

thatatb6expressionincoloncancercellsleadtotheincreasein

secretionofMMP-9andthatthissecretionparalleledthelevelof

cell-surfaceatb6expression.13Inourstudy,tofurtherinvestigate

themechanismbetweenatb6andECMdegradationweused

RNAinterferingtechnologytosuppresstheexpressionofatb6in

coloncancercells.WetransfectedHT29cellswithaplasmidin

which,weclonedDNAfragmentsthatactedastemplatesforthe

synthesisofsiRNAsunderthecontroloftheU6promoter.The

resultsobtainedconfirmanddemonstratethatdownregulationof

atb6hasdecreasedthelevelsofthesecretionofuPA,pro-MMP-

9andpro-MMP-2intumorconditionedmedium(TCM)and

inhibitedtheMAPKdependentECMdegradationthroughthe

plasminogenactivationcascade.

Materialandmethods

Antibodiesandreagents

Themonoclonalantibodyagainstatb6(R6G9,10D5)were

fromChemiconInternational.Themonoclonalantibodyagainst

ERK1/2andPhospho-ERK1/2werefromCellSignalingTechnol-

ogy,Inc.Anti-actinandanti-GAPDHwaspurchasedfromCalbio-

chem.Amiloride(uPAinhibitor),1,10-phenanthroline(anti-MMPs)

andMEK1inhibitorU0126,horseradishperoxidase(HRP)-la-

beledsecondaryantibodywerefromPromega.

SelectionofsiRNAtargetingsitesandplasmidconstruction

WefollowedthecriteriadescribedbyReynoldsetal.14and

usedahelpfulwebbasedtool(http://www.dharmacon.com)to

designsiRNAstargetingagainstb6codingregionbasedonhuman

integrinsubunitbeta6cDNA(GenBankaccessionnumbers:

A266609).Theselectedsequencewasscreenedagainstthehuman

genomebyusingaBLASTsearchtoavoidunintentionalsilencing

andensurethatonly1humangenewastargeted.Thedouble-

strandedoligo-DNA(dsDNA)usedinourstudywereasfollows:

sense(50-GATCCCCTACAGTTAATTTGAAGTTTCAAGA

GAACTTCAAATTAACTGTAGGTTTTTTG-30),antisense(50-

AATTCAAAAAAACTTCAAATTAACTGTAGGTCTCTTGAA

CCTACAGTTAATTTGAAGTG-30),andthissequence(ACTT-

CAAATTAACTGTAGG)correspondedtonucleotides431–449

ofthehumanb6cDNA.Thepairofcomplementaryoligonucleo-

tideswithaBamHIsiteandEcoRIsiteaddedtotheir50-and30-

ends,respectively,weresynthesized(Invitrogen,Shanghai,

China).TheresultingdsDNAwhichwascomposedof2identical

Abbreviation:dsRNA,doublestrandedRNA;ECM,extracellularma-

trix;MAPK,mitogen-activatedproteinkinase;RNAi,RNAinterference;

siRNA,smallinterferingRNA;TCM,tumorconditionedmedium;uPA,

urokinaseplasminogenactivator.

Grantsponsor:NationalNaturalSciencesFoundationofChina;Grant

number:30570833;Grantsponsor:NaturalSciencesFoundationofShan-

dongProvince;Grantnumber:Y2005C42.

Thefirsttwoauthorscontributedequallytothiswork.

*Correspondenceto:DepartmentofGeneralSurgery,QiLuHospital,

AffiliatedtoShandongUniversity,Jinan250012,Shandong,China.Fax:

186-0531-82169203.E-mail:junniu120@sina.com.cn

Received9March2008;Acceptedafterrevision26March2008

DOI10.1002/ijc.23656

Publishedonline19June2008inWileyInterScience(www.interscience.

wiley.com).

Int.J.Cancer:123,1311–1317(2008)

'2008Wiley-Liss,Inc.

PublicationoftheInternationalUnionAgainstCancer


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