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miR-448通过抑制上皮间质转化抑制肺癌细胞增殖和运动能力
优质期刊推荐Hepatic Premalignant Alterations Triggered by Human Nephrotoxin Aristolochic Acid I in Canines_图文-五星文库
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Hepatic Premalignant Alterations Triggered by Human Nephrotoxin Aristolochic Acid I in Canines_图文
导读：PublishedOnlineFirstFebruary5,2016;DOI:10.07.CAPR-15-0339ResearchArticleCancerPreventionResearchHepaticPremalignantAlterationsTriggeredbyHumanNephrotoxinAristolochicAci
Published OnlineFirst February 5, 2016; DOI: 10.07.CAPR-15-0339
ResearchArticle
CancerPreventionResearch
HepaticPremalignantAlterationsTriggeredbyHumanNephrotoxinAristolochicAcidIinCanines
KeJin1,Kun-kaiSu2,3,TongLi1,Xia-qingZhu1,QiWang1,Ren-shanGe4,5,Zong-fuPan1,Bo-wenWu1,Li-junGe1,Yi-hanZhang1,Yi-fanWang1,Guo-fangShen1,Dan-yanZhu1,Chun-shengXiang2,3,Lan-juanLi2,3,andYi-jiaLou1
Introduction
Hepatocellularcarcinoma(HCC)isoneofthemostlethalmalignanciesandthefourthmostcommoncancerworldwide,witharound700,000newcaseseachyear(1,2).Environmentalfactorsincludingdietaryhabits,lifestylechoices,andevendrugtherapeuticsplayimportantrolesinmosthumancancers(3C5),includingexcessivealcoholconsumptioncorrespondingtoHCC(6,7).PlantdrugsderivedfromAristolochiaspp.remaininuse
InstituteofPharmacologyandToxicology,CollegeofPharmaceuticalSciences,ZhejiangUniversity,Hangzhou,PRChina.2StateKeyLabo-ratoryforDiagnosisandTreatmentofInfectiousDiseases,Hangzhou,PRChina.3CollaborativeInnovationCenterforDiagnosisandTreat-mentofInfectiousDiseases,The1stAf?liatedHospital,CollegeofMedicine,ZhejiangUniversity,Hangzhou,PRChina.4ThePopulationCouncilattheRockefellerUniversity,NewYork,USA.5InstituteofReproductiveBiomedicine,the2ndAf?liatedHospital,WenzhouMed-icalUniversity,Wenzhou,PRChina.
Note:SupplementarydataforthisarticleareavailableatCancerPreventionResearchOnline(http://cancerprevres.aacrjournals.org/).K.JinandK.-k.Sucontributedequallytothisarticle.
CorrespondingAuthors:Yi-jiaLou,InstituteofPharmacologyandToxicology,CollegeofPharmaceuticalSciences,ZhejiangUniversity,866YuhangtangRoad,Hangzhou310058,PRChina.Phone:86-571-;Fax86-571-;E-mail:yijialou@;Lan-juanLi,The1stAf?liatedHospital,CollegeofMedicine,ZhejiangUniversity,79QingchunRoad,Hangzhou310003,PRChina.E-mail:ljli@
doi:10.07.CAPR-15-0339
ó2016AmericanAssociationforCancerResearch.
todayforthetreatmentsofsnakebites,arthritisandgout,andcoronaryarterydiseases(8,9),particularlyinAsiaandsomeotherareas,withthepotentialforfurtherexposure(10).Aristolochiaherbscreateapotentialpublichealthproblemofconsiderablemagnitude(11).Aristolochicacid(AA),anactivemixtureofAAIandAAII,existedinplantdrugsfromAristolochiaspecies.AAI,proventobethecauseofAAnephropathyinwomen,wasassociatedwithprolongedintakeofChineseherbalremediescontainingAAIindietarysupplementsforslimmingforthe?rsttimeinBelgiumin1991(12).Thepatientshaddevelopedahighriskofuppertracturothelialcarcinoma(about50%)and,sub-sequently,bladderurothelialcarcinoma(13,14).AAIcanalsoinducetumorsinmultipleorgansofmicewithin56weeksafterthestartofa3-weektreatment(15).Notably,NF-kB1andc-Myconcoproteinexpressioninkidneyreachapeakat12-dayAAIadministrationofHupki(humanTP53knock-in)mice(16,17),suggestingacriticalroleofrenaltumorigenesisintheshort-termdurationofAAI(16,17).Geneassociationnetwork(GAN)anal-ysisfurthersuggestshepatocytenuclearfactor4agene(Hnf4a)andTp53inhibitioninAAI-treatedHupkimouseliver(DNArefs.16,17).AA-likemutationalsignaturesin11HCCgenomes/exomeshavealsobeenreported(18).TransientinhibitionofHNF4ainitiateshepatocellulartransformationthroughamicroRNA(miRNA)in?ammatoryfeedbackloopcircuit(19);however,whethershort-termAAIexposureinducespotentialhepatocarcinogenesisisnotcon?rmedsofar.
Short-termAAIexposuremarkedlyupregulatesc-Myconco-proteinexpressioninthekidneyofHupkimice(16).Overexpres-sionofc-MyccaninduceHCCwithahighfrequencyinmice(20).
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AAI-InducedHepaticPremalignantAlterations
Activationofc-Myconcogenictranscriptionfactor(Myc)inducesmeasurement.Caninesweresacri?ced11daysafterinitiationtheoncofetalRNA-bindingproteinLin28Bexpressioninmultipleofthetreatment.Liverswereexcisedimmediatelyaftersacri?ce.humanandmousetumormodels(21).Theoutcomesubsequent-Partoftheliverwas?xedin4%(wt/vol)neutralbufferedlyinhibitsthebiogenesisofalllet-7miRNAs,therebypromotingformalin(pH7.4)andembeddedinparaf?nforhistologicembryo,stemcell,andtumorgrowth(22).Infact,upto40%theremainingliverwasimmediatelysnap-frozeninHCCsareclonalandthusareconsideredtooriginatefromhepaticliquidnitrogenandkeptatà80 Cuntiluse.
progenitorcells(HPC;refs.23,24).WithinhumanHCC,cellshavingsignaltransducersandactivatorsoftranscription3Histologicevaluation
(STAT3),stemnessfactorOct4butlackingtheTGFbreceptortypeLiversectionswereembeddedinparaf?nandpreparedII(TBRII)andembryonicliverfodrin(ELF)arebelievedtobeaccordingtotheprotocol(16).Liversectionswerestainedwithtypicalhepaticcancerprogenitor-likecells(HCPLC;refs.23,24).hematoxylinandeosin(H&E)toevaluatehistologicdamage.Todate,however,whethertheinitialmoleculareventsandImmunohistochemicalanalysiswasperformedaccordingtothesignalingtransductionareinvolvedintheonsetandprogressionprotocol(2).Typically,mousemonoclonalantibodyagainstofmalignantHPCsisstillnotcon?rmed.BasedonthefactthatSTAT3(CellSignalingTechnology,#),rabbitplantdrugsderivedfromAristolochiaspp.remaininuseevenmonoclonalantibodyagainstp-STAT3(Tyr705,CellSignalingtoday,itisparticularlyimportanttoexplorethemostrelevantTechnology,#),rabbitpolyclonalantibodyagainstnodesofcross-talkandcooperativityofthesignalmoleculesinthep-FOXO1(Ser256,Abcam,ab:100),rabbitpolyclon-initialstageofHCPLCformationbyAAIexposure.
alantibodyagainstOct4(Abcam,ab),andrabbitMiRNAs,akindof20C23-ntnoncodingfunctionalRNAmole-polyclonalantibodyagainstIL6Ra(SantaCruz,sc-13947,cules,arecriticaldownstreamcomponentsofkeyoncogenicand1:100)wereincubatedwithliversectionsrespectivelyat4 Ctumorsuppressorsignalingpathways(25C27).Directcontrolofovernightsubsequently.ThefollowingstainingweredevelopedmiRNAexpressionbyoncogenicandtumorsuppressornetworkswithanimmunohistochemicalstainingkit(SA1022orSA1021,resultsinfrequentdysregulationofmiRNAs,whichcontributestoBoster)andaDABkit(AR1022,Boster)accordingtothetumorigenesis(21).Short-termAAIexposurealsocausesNF-kB1instructions.
overexpressioninthekidneyofHupkimice(16).IntheimmuneForimmuno?uorescentanalysis,rabbitmonoclonalantibodysystem,enhancedregulationofNF-kBsignalingiscrucialforagainstc-Myc(Abcam,ab),rabbitpolyclonalanti-maintainingthenormalfunctionofimmunecellsandavoidancebodyagainstLin28B(SantaCruzBiotechnology,sc-130802,oftumorigenesis(28,29).AlthoughtheimportanceofmiRNAsin1:100),mousemonoclonalantibodyagainstOct4(Abcam,thesignaltransductionofIL6andNF-kBhasbeenhighlightedinab),rabbitmonoclonalantibodyagainstSTAT3(CellhumanHCC(19,28,30)andinhumanacute-phaseresponseofSignalingTechnology,#0),rabbitpolyclonalanti-hepatocytes(31),todate,whetherIL6R/NF-kBcontributestobodyagainstTBRII(SantaCruzBiotechnology,sc-400,1:100),miRNAdysregulation,therebycausingpremalignanceinliverbyrabbitpolyclonalantibodyagainstOct4(Abcam,ab18976,short-termAAIexposurehasnotyetbeenillustrated.
1:100),mousemonoclonalantibodyagainstELF(344050,Inthepresentresearch,wefoundthatc-MycandLin28Bwere1:100,MerckMillipore)wereincubatedwithliversectionsat4 CoverexpressedintheAAI-inducedacute-phasehepaticpatho-overnightfollowedbyincubationwithDylight488or549Clogicalresponseincanines.Yet,AAIexposureenhancedtheconjugatedgoatanti-mouse(GAM4882orGAM),vulnerabilityofHPCstoHCPLCs.IL6R/NF-kBactivationlink-goatanti-rabbit(GAR4882orGAR)secondaryanti-ingdysregulatedmiRNAswasinvolvedinnodesofhepaticbodies(Multisciences).
premalignantalteration.Itshedsanewinsightintotheinter-ForTUNELstainingassays,liversectionswerestainedusinganplayofabnormalsignalingactivationintheAAI-inducedInSituCellDeathDetectionKit(,RocheDiagnos-premalignantprocessandsuggeststhatanti-premalignantther-tics)followingthemanufacturer'sinstructions.
apymaybeanadmissiblestrategyforpreventinglivertumor-ThesampleswereanalyzedwithaLeicaphase-contrastmicro-igenesiscausedbyAAIexposure.
scope(DMI3000B)forimmunohistochemicalanalysisandOlympus(BX61W1-FV1000)confocalmicroscopeforimmuno-MaterialsandMethods
?uorescentanalysis.DigitalimageswereanalyzedusingLeicaApplicationSuitev4.2andOlympusFluoviewFV1000,andAnimalsandanimalworks
?gureswerepreparedbyusingMacromediaFireworksv8.0.Ten-month-oldmalebeaglecanineswerepurchasedfromtheANNIMOScienceandTechnologyLtd[Certi?cateNo.SCXK(Su)Liversamples'molecularandbiochemistryanalysis
]andweremaintainedinaspeci?cpathogen-freeELISAassaysusingthecanineIL6ELISAkit(CA6000;R&Denvironment.TheproceduresforperforminganimalexperimentsSystems)wereperformedusingtheDTX880MultimodeDetectorwereinaccordancewiththeprinciplesandguidelinesofZhejiang(BeckmanCoulter).
Universityforanimaluse.AndaprotocolwasapprovedbytheWesternblotandcoimmunoprecipitationanalyseswereper-InstitutionalAnimalCareandUseCommitteeofZhejiangUni-formedaccordingtotheprotocol(16).Brie?y,livertissueswereversity,China(EthicsCode:No.zjuY).
homogenizedwithRIPAbuffer.NuclearandcytoplasmicproteinsAAI(purity&98%,HPLC,Delta)wasmixedwith?llerandwereseparatelyisolatedusinganuclearextractionkit(P0028,?lledintocapsules.CanineswererandomlyassignedtotwoBeyotime).Thefollowingprimaryantibodieswereused:Smad2/3groups(4ineachgroup)andgivencapsuleswithcontrol?ller(#,000),Smad4(#,000),p-IkBa(Ser32,orAAI?ller(3mg/kg/day,equivalentdoseofmouse)for10#,000),NF-kBp50(#,000),p65(#4764,days.Bloodsampleswerecollectedatindicateddaysforserum1:2,000),c-Fos(#,000),c-Jun(#,000),EGFRalaninetransaminase(ALT)andaspartatetransaminase(AST)
(#,000),p-EGFR(Tyr,1:1,000),STAT3
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(#,000),p-STAT3(Tyr705,#,000),Januskinase2(JAK2;#,000),Akt(#,000),p-Akt(Ser473,#,000),andp-Akt(Thr308,#,000;CellSignalingTechnology);Lin28B(sc-:200),TBRII(sc-400,1:200),p-Smad2/3(Ser423/425,sc-),IL6Ra(sc-),andLaminB(sc-:100;SantaCruzBiotechnology);Oct4(ab),TGFb1(ab),p-TBRI(Ser165,ab:200),c-Myc(ab,000),FOXO1(ab:500),andp-FOXO1(Ser256,ab:500;Abcam,USA);GAPDH(Mab,Multi-sciences).OtherinformationaboutprimaryantibodiesusedinSupplementaryFig.S1isgiveninSupplementaryTableS3.TheHRP-conjugatedsecondaryantibodiesweregoatanti-mouse(LK-GAM007),goatanti-rabbit(LK-GAR007),andrabbitanti-goat(LK-RAG007,Multisciences).Coimmunoprecipitationwasperformedforanti-STAT3andOct4accordingtotheprotocol(32).
RT-PCRanalysis
RT-PCRanalysiswasconductedaccordingtotheprotocol(33).Brie?y,totalRNAsfromlivertissueswereisolatedwithTrizolreagent(GibcoBRL).ThemRNAexpressionlevelswerenormal-izedtotheexpressionofthehousekeepinggeneGapdh.TheprimersequencesandconditionsarepresentedinSupplementaryTableS1.
MiRNAmicroarrayandtargetprediction
MiRNAmicroarrayanalysiswasperformedbyKangChenBio-tech(Shanghai)withExiqonmiRCURYLNA,whichisavailableatGSE75474(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc?GSE75474).MiRNAsdysregulated&1.5-fold(P&0.05)inresponsetoAAIwerefurtherevaluatedusingqRT-PCRbyKangChenBio-tech.MiRNAclusterswereretrievedfrommiRBase(www.mirbase.ref.34).PutativetargetsofmiRNAswerepredictedbythecombinationofTargetScan(www.targetscan.org)andmiRDB(www.mirdb.refs.35,36).Finally,theestimatedtargetproteinexpres-sionwascon?rmedbyWesternblotanalysis.
CellcultureandmiRNAtransfection
ThehumanHCCcelllineHepG2waspurchasedfromtheShanghaiInstituteofCellBank,ChineseAcademyofSciences(purchaseordernumberNo.22008),andthecelllinewasauthenticatedbypassingtheconventionaltestsofcelllinequalitycontrolmethodsandthetestofDNApro?ling(STR).CellsweremaintainedinhighglucoseDMEM(,LifeTechnolo-gies)supplementedwith10%fetalbovineserum(FBS;Gibco,LifeTechnologies).Thecellsweretransfectedwithlet-7bmimicorinhibitor,miR-27amimicorinhibitorbyLipofectamine-019,Invitrogen,LifeTechnologies),respectively,for24hours,thentreatedwithorwithoutIL6(20ng/mL,#200-06,PeproTech)foranother24hours.Cellswerecollectedafter48hours.ThesequencesofmiRNAmimicandinhibitorareshowninSupplementaryTableS2.
Statisticalanalysis
Pairwisecomparisonsbetweencontinuousdatawereana-lyzedusinganunpairedtwo-tailedStudentttest,andmultiplecomparisonswereanalyzedbyone-wayANOVA.Alldatawereexpressedasmean?SD,andP&0.05wereconsidered
statisticallysigni?cant.ThebioinformaticsdatawereanalyzedbytheMannCWhitneyUtest,andP&0.05wasconsideredstatisticallysigni?cant.
AAItreatmentcausesliverinjuryrelatedtoapoptosis
AsshowninFig.1A,AAI-treatedliversectionsdisplayedcongestionwithdisorderstructureandkaryopyknosis.Mean-while,TUNELstainingrevealedanincreasednumberofapo-ptoticcells(P&0.01)intheliversectionofAAI-treatedcanines(Fig.1B).SerumALTandASTlevelsshowedtime-dependentincreases,indicatingthatliverfunctionaldamagewasprogres-sing.ALTlevelswereincreasedatday5andmarkedlyelevated(by20-fold,P&0.01)atday9intheAAI-treatedcanineswhencomparedwiththoseofcontrol.SimilarresultswereobservedforASTlevels(Fig.1C).
AAItreatmentleadstoc-MycandLin28Bupregulation
Insitudouble-stainingimmuno?uorescenceexhibitedtheover-expressionofc-MycandLin28BintheliversectionsreceivingAAI(Fig.2A).Westernblotanalysisfurthershowedthattheirexpres-sionswereupregulatedby2.5-foldintheAAI-treatedlivercom-paredwithcontrol(P&0.05,Fig.2B).Inaddition,theexpressionlevelsofHCCgenea-Fetoprotein(Afp)andAFPproteinwereincreasedandreached10-foldor5-foldhigherthanthoseincontrol,respectively(P&0.01,SupplementaryFig.S1),implyingthatthelivermightundergopremalignantlesioninresponsetoAAIadministration.Theseresultsrevealedthatshort-termAAIexposurecouldleadtohepaticpremalignantalterationsincaninesasearlyasorallyadministratedfor10days.
AAItreatmentinducesHCPLCappearance
Here,weusedtheimmunohistochemistry,Westernblot,andcoimmunoprecipitationassaystorevealtheexpressionofOct4anditsbindingtoSTAT3intheliversofcaninesreceivingAAI.Asaresult,Oct4-positivecellsemergedasclustersinresponsetoAAI(Fig.3A).Comparedwithnegativecontrol,theOct4proteinlevelwassigni?cantlyincreased(P&0.01,Fig.3B),andboundtoSTAT3inAAI-treatedliver(Fig.3C).Furthermore,insitudouble-stainingimmuno?uorescencedemonstratedthatsomeHPCswiththetypicalmarkersOct4,STAT3,TBRII,andELFappearedinliversectionsbyAAIexposure(Fig.3D),implyingthatHPCsprolif-eratedandmigratedinresponsetoAAI-inducedapoptosis.Importantly,amongthem,afewcellsco-labeledwithOct4andSTAT3,butlackingbothTBRIIandELF,appearedinparallel(Fig.3D),suggestingthatpremalignantalterationwithatypicalfeatureofHCPLCsformedintheliverreceivingAAI.ConsideringthatenhancedTGFbsignalingmaypromotedevelopmentofHPCs(37),wefurtherexaminedwhetherAAItreatmentactivatedtheTGFb1/Smadpathway.AsshowninFig.3E,thedownstreameventsofTGFb1,suchasp-TBRI,p-Smad2/3(includingcyto-plasmandnucleus),andSmad4,werephosphorylatedorover-expressed.Unexpectedly,TGFb1/SmadpathwayactivationdidnotaffecttheE-cadherinexpressionasreported(ref.38Supple-mentaryFig.S2).
AAI-inducedhepaticmiRNAdysregulationandrelatedtargetvalidation
WenextobservedthedysregulatedmiRNAsandtherelatednetworksintheliverreceivingAAI.Theheatmaprepresents
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AAI-InducedHepaticPremalignantAlterations
LiverinjuryaccompaniedbyapoptosisbyAAIexposureincanines.A,H&Estainingshowedthelesiontissue(arrowheads);B,TUNELstainingdisplayedapoptoticcells
(arrowheads)inliversectionsfromAAI-treatedcanines.Thetotalnumberofapoptoticcellswasnormalizedtothecontrolinareaofthetissueslice
(cells/mm2
).C,elevatedserumALTandASTdemonstratedliverfunctionaldamageinatime-dependentmanner.Errorbars,meanvalue?SD;?,P&0.05,??
,P&0.01versuscontrol.Foreachanalysis,n?4pergroup.
miRNAmicroarrayanalysisofliverbyAAIexposurecomparedand2.0Cfold,respectively(P&0.01),whereasmiR-27awaswithnegativecontrol(SupplementaryFig.S3).Especially,10increasedby1.95-foldandmiR-24by2.5-fold(P&0.01,Fig.4B).miRNAswerepredominantlydifferentiallyexpressedandiden-GiventhatdysregulatedmiRNAspossesstheenhancedpost-ti?edinliverbyAAIexposurecomparedwiththeliverintranscriptionalregulationintheirtargets,wethereforeevalu-control(P&0.05,P&0.01,Fig.4AandB).Amongthem,atedthepossibleeffectsofAAIexposureonputativetargetsofcfa-let-7a-1$let-7bmiRNAsinthe10q12.3chromosomelet-7a-1$let-7bormiR-27a$miR-24accordingtotheputativeregionandcfa-miR-27a$miR-24inthe20q13.2chromosometargetintersectionsbetweenTargetScanandmiRDB(Supple-region(SupplementaryFig.S4)werecloselylinkedtotumor-mentaryFig.S4).Westernblotresultsshowedthevalidationigenesisorpremalignantlesion.TheqRT-PCRassayfurtherresultsofputativepotentialtargetsmentionedabove(Supple-con?rmedthatlet-7a-1andlet-7bweredecreasedby3.3-foldmentaryFig.
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MicroRNA—29a 在脂多糖诱导人单核细胞凋亡中的作用和机制
　　【摘要】目的 探讨microRNA-29a（miR-29a）对人单核细胞株THP-1凋亡的作用及其机制。方法 体外培养人单核细胞株THP-1和人胚肾细胞株293T，合成人miR-29a的拟似物（mimic）和抑制剂（inhibitor）。用脂质体Lipofectamine RNAiMAX转染miR-29a的mimic或inhibitor进入THP-1细胞，分组处理后收集细胞标本。第1组细胞转染mimic （100 nmol/L） 48 h；第2组细胞先转染 inhibitor （100 nmol/L） 24 h，再用脂多糖（LPS） 诱导24 h，分别用流式细胞仪方法检测两组细胞凋亡，用real time RT-PCR 方法检测抗凋亡基因 Bcl-2和Mcl-1 的表达变化。构建Bcl-2 和Mcl-1 的荧光素酶报告基因载体，用脂质体 Lipofectamine 2000 转染293T 细胞（DNA 质粒和miRNA 片段共转染），双荧光素酶报告基因系统（luciferase）检测荧光素酶的表达变化。应用SPSS 13.0统计软件，采取单因素方差分析或t检验进行数据统计分析。结果 THP-1细胞转染miR-29a 的mimic 48 h后，细胞凋亡较对照组增加（17.38%增加至42.06%）；单独用LPS 诱导THP-1 细胞，24 h 后细胞凋亡较对照组增加；THP-1 细胞先转染inhibitor 24 h 后，再用LPS诱导24 h，细胞凋亡较单独用LPS诱导组减少（由51.50%降至38.09%）；THP-1细胞转染miR-29a的mimic后，抗凋亡基因Bcl-2和Mcl-1的表达水平降低明显（P<0.05）。另外，Luciferase 检测结果显示，在293T细胞中，双萤光报告系统显示 miR-29a可特异抑制带有 Bcl-2 和Mcl-1 3’UTR上野生型识别元件的报告基因表达（P<0.05）。结论 上调miR-29a的表达水平能促进THP-1细胞发生凋亡，下调miR-29a的表达水平则能抑制LPS诱导的THP-1细胞凋亡，miR-29a调控THP-1的凋亡水平是通过靶向于两个抗凋亡基因Bcl-2和Mcl-1实现的，提示miR-29a在调控免疫细胞的凋亡过程中具有重要的作用。 中国论文网 /6/view-3913911.htm　　【关键词】微小RNA；单核细胞；脂多糖；凋亡；Bcl-2基因；Mcl-1基因 　　MicroRNA-29a regulates apoptosis induced by lipopolysaccharide in THP-1 cells XIONG Xu-ming， ZHANG Zhen-hui， JIANG Zi-xin， CHEN Wei-yan， YANG Qi-lin， LIU Wei-jiang. Intensive Care Unit， The Second Affiliated Hospital of Guangzhou Medical College， Guangzhou 510260， China 　　【Abstract】Objective To investigate the effects of microRNA-29a （miR-29a） on lipopolysaccharide （LPS）-induced apoptosis in human monocytes THP-1 cells in order to understand the molecular mechanisms. Methods Human monocytes THP-1 cell line were exposed to LPS after transfected with miR-29a inhibitors （100 nmol/L） or just transfected with miR-29a mimic （100 nmol/L） by lipofectamine RNAiMAX. Flow cytometry （FCM） was used to detect the cell apoptosis. Real-time RT-PCR was employed to measure expressive levels of the gene Bcl-2 and Mcl-1. The luciferase assay was performed in HEK293T cells， which were co-transfected with plasmid DNA and miRNA by using Lipofectamine 2000. Statistical analysis carried out by using SPSS 13.0 software for One-way ANOVA and Student’s t test. Results Transfection with miR-29a mimics for 48 h increased apoptosis rate and significantly reduced the expressions of Bcl-2 and Mcl-1 in THP-1 cells in comparsion with the control group. The apoptosis rate also raised in THP-1 cell stimulated by LPS for 24 h followed by LPS stimulation for 24 h， the apoptosis rate was decreased in comparison with the LPS group. In addition， our luciferase assay data showed that HEK293T cells co-transfected with miR-29a mimics and Bcl-2 3’UTR-Wt or Mcl-1 3’UTR-Wt plasmid significantly reduced the luciferase activity compared with the control group. Conclusions The miR-29a may regulate apoptosis by targeting the genes Bcl-2 and Mcl-1， and miR-29a may play a pivotal role in the process of apoptosis in immune cells. 　　【Key words】MicroRNA；Monocytes；Lipopolysaccharides；Apoptosis；Bcl-2；Mcl-1 　　脓毒症（sepsis）具有患病率高、病死率高、治疗费用高等特点［1-3］，是目前重症医学领域最受关注的临床难题之一。而脓毒症的发病机制仍欠清晰，免疫功能紊乱尤其是免疫功能抑制被认为是脓毒症发病的重要环节，其中免疫细胞凋亡与免疫功能抑制密切相关，抑制免疫细胞凋亡能有助于改善机体免疫功能，进而提高生存率［4］。范震等［5］的研究结果显示microRNAs（miRNAs） 参与脓毒症的发病过程。有研究结果表明，microRNA-29a （miR-29a）参与肿瘤细胞凋亡的调控，而关于miR-29a对免疫细胞状态的影响及其机制的研究，目前仍缺乏报道。本研究采用内毒素脂多糖（LPS）诱导人单核细胞株THP-1，模拟脓毒症免疫细胞炎症反应过程，并通过细胞转染的方法，将 miR-29a的拟似物（mimic）或抑制剂（inhibitor）导入细胞内，上调或下调细胞内miR-29a的表达水平，进而观察细胞凋亡水平的变化并探讨其相关分子机制，为阐明miRNAs在脓毒症中的作用及寻找脓毒症干预的新靶向提供实验依据。 　　1 材料与方法 　　1.1 材料 　　人单核细胞系（THP-1）细胞株和人胚肾细胞株293T（购自上海细胞库）。内毒素（LPS）（购自美国Sigma公司）；RPMI-1640培养基、DMEM培养基、Opti-MEM培养基、胎牛血清、β-巯基乙醇（购自美国GIBCO公司）；Lipofectamine RNAiMAX、Lipofectamine 2000、Trizol、SYBR Green qPCR 试剂盒均购自Invitrogen公司； Annexin V-FITC 凋亡检测试剂盒（购自南京凯基生物公司）；报告基因活性检测试剂盒（Dual-Luciferase? Reporter Assay System， Promega）；pGL3-control-MCS和Renilla luciferase质粒（Invitrogen公司），内切酶和连接酶（美国Fermentas公司），质粒提取试剂盒（美国Omega）。 　　1.2 方法 　　1.2.1 细胞培养及传代方法 将THP-1细胞置于含10％胎牛血清的RPMI1640培养基中，于37 ℃、5％ CO2培养箱静置培养。该细胞株属人类单核细胞系，在培养液中呈簇集状悬浮生长，细胞含量控制在1×106/ml，每2 d换液一次，每4 d传代一次。细胞复苏后，传至3～5代后用于实验。细胞用含10％胎牛血清的DMEM培养基培养，待细胞贴壁融合率达到80%时进行转染。 　　1.2.2 实验分组 将按1.2.1项操作所得THP-1细胞分为正常对照组（CON组）、LPS组（加入LPS 1 μg/ml诱导细胞，相应CON组用PBS代替LPS，诱导24 h后收集细胞检测）、miR-29a拟似物转染组（简称miR-29a mimic组，转染48 h后收集细胞检测），miR-29a拟似物转染对照组（negative control组，简称NC组，转染48 h后收集细胞检测），miR-29a抑制剂转染组（简称miR-29a inhibitor组，转染48 h后收集细胞检测）。 　　1.2.3 寡核苷酸序列设计与合成 miR-29a的拟似物（mimic）根据其成熟体序列（miRBase accession No.MI0000087）合成。miR-29a的抑制剂则采用 miR-29a成熟体的反向互补序列，并对所有碱基都进行 2’-甲氧修饰（由吉玛公司设计）。双链 RNA 的负对照（NC）为与哺乳动物基因组没有同源性的序列，该序列的设计和所有核苷酸的合成均由上海吉玛公司完成。所有PCR引物均由上海英骏生物公司合成（表1）。 　　1.2.4 载体构建 Bcl-2 和 Mcl-1 3’UTR 报告基因载体 pGL3-BCL2-3’UTR-Wt 和pGL3-MCL1-3’UTR-Wt 携带野生型（wild type， Wt）的 3’UTR，而 pGL3-BCL2-3’UTR-Mut 和pGL3-MCL1-3’UTR-Mut 则携带突变了 miR-29 识别元件的 3’UTR（突变体，Mutation， Mut）。所有载体均以 pGL3-control-MCS为基础，在 ApaI 和 XbaI 的酶切位点间插入野生型或突变型的 Bcl-2 和 Mcl-1 3’UTR 序列获得的。野生型插入片段的模板是 BCL2 基因（NM_000633）和 MCL1 基因（NM_021960）的 3’UTR。突变型插入片段的模板是以野生型载体为基础，在 PCR 扩增使用的引物上引入突变位点，在分别扩增突变位点上下游两段序列后，通过融合 PCR及酶切产生［6］。 　　1.2.5 细胞转染和报告基因活性检测 miRNA的单独转染采用转染试剂Lipofectamine RNAiMAX。以6孔板为例，THP-1细胞更换新鲜的RPMI 1640培养基（不含FBS和抗生素）每孔2 ml。将miRNA稀释于200 μl Opti-MEM培养基中；每管加入8 μl Lipofectamine RNAi MAX，轻柔混匀，室温放置15 min；将 RNAi MAX与siRNA的稀释液加入 6孔板的各孔细胞中，混匀后培养箱中培养48 h，48 h后按实验需要处理细胞。miRNA的终浓度为100 nmol/L。转染后24 h加LPS，加LPS后24 h收集样本。 　　在双荧光素酶报告系统（luciferase）实验中转染 293T 细胞：首先在转染前一天，细胞先传代铺于48 孔板培养（用不含FBS和抗生素的DMEM培养基），铺板后24 h，用Lipofectamine 2000 进行转染，将小分子 RNA 和表达质粒稀释于25 μl opti-MEM 中，轻柔混匀；将1 μl Lipofectamine 2000稀释于25 μl opti-MEM 中，混匀，室温放置 5 min；将上两液混合，室温放置 20 min；然后加入 48孔板各孔的细胞中， RNA的浓度为 10 nmol/L， firefly luciferase质粒10 ng，Renilla luciferase 质粒 2 ng，混匀后培养箱中培养6 h，更换培养基（含10%FBS的DMEM）再培养42 h后收集细胞，按照报告基因活性检测试剂盒按说明书，用化学发光法检测酶活性。 　　1.2.6 RNA提取及real time RT-PCR 细胞总 RNA 提取按 Trizol 试剂盒说明书操作；使用invitrogen的逆转录反应试剂盒，按试剂盒说明书进行，反应产物置于-20 ℃保存备用或立即进行PCR扩增；PCR过程按invitrogen的SYBR Green qPCR 试剂盒说明书在ABI 7500荧光定量PCR仪上进行，使用18S作为内参照同时进行PCR，获得Ct值后，应用比较Ct法进行相对定量，目标基因的相对定量用2-△△Ct计算。 　　1.2.7 细胞凋亡水平检测（Annexin V-FITC 染色法） 收集细胞时直接将悬浮生长的THP-1细胞轻轻吹打混匀，收集细胞悬液，500 r/min 4 ℃离心10 min，弃上清液。将细胞悬于标记缓冲液中。然后，加入 Annexin V-FITC，避光室温反应 15 min。加入标记缓冲液，用流式细胞仪观察细胞凋亡情况。 　　1.3 统计学方法 　　采用SPSS 13.0 统计软件进行数据的分析处理，计量资料采用均数±标准差（x±s）表示，组间差异只有两组时用成组t检验，有多组时用单因素方差分析（One-Way ANOVA）。以P<0.05为差异具有统计学意义。 　　2 结果 　　2.1 过表达miR-29a促进THP-1细胞凋亡 　　THP-1细胞转染miR-29a mimic 100 nmol/L后，经流式细胞仪检测，与NC组比较，细胞存活率由82.62%下降至57.94%，提示THP-1细胞过表达miR-29a可导致细胞凋亡水平增加（图1）。 　　2.2 下调miR-29a的表达水平能抑制LPS诱导的THP-1细胞凋亡 　　THP-1细胞先转染miR-29a 的抑制剂100 nmol/L，24 h后再用LPS（1 μg/ml）诱导，与单纯用LPS（1 μg/ml）诱导比较，抑制剂组细胞的存活率增高（由48.50%提高至61.91%），提示下调THP-1细胞miR-29a的表达水平可抑制LPS诱导的细胞凋亡（图2）。 　　2.3 过表达miR-29a后 Bcl-2 和Mcl-1的表达下降 　　THP-1细胞转染miR-29a 100 nmol/L 48 h后，用real time RT-PCR检测Bcl-2 和Mcl-1 mRNA的表达水平，结果提示，与CON组和NC组比较，Bcl-2 和Mcl-1的mRNA表达水平均下降，降至（0.55±0.12）和（0.65±0.06），P<0.05，提示过表达miR-29a，能使 Bcl-2 和Mcl-1的表达水平下降（图3）。 　　2.4 双荧光素酶报告基因系统验证miR-29a的靶基因 　　293T细胞同时转染miR-29a的mimics和Bcl-2 （或Mcl-1）的报告基因载体（Wt野生型和Mut突变体），结果显示，与对照组比较，转染Bcl-2或Mcl-1野生型（Wt）载体的细胞荧光素酶活性下降，分别由（1.07±0.18）和（1.00±0.03）降至（0.63±0.12）和（0.58±0.10），P0.05）。结果提示miR-29a能特异性地与Bcl-2和Mcl-1基因的3’UTR的种子序列结合，降解Bcl-2和Mcl-1的mRNA（图4）。 　　3 讨论 　　脓毒症的发病机制异常复杂，涉及感染、炎症、免疫、凝血、细胞凋亡以及神经-内分泌异常等一系列问题，并与机体多系统、多器官病理生理改变密切相关［7］。免疫功能紊乱尤其是免疫功能抑制被认为是脓毒症发病的重要环节，其中免疫细胞凋亡是脓毒症免疫抑制状态发生的一个重要机制，直接影响脓毒症患者的转归和预后［8］，可能为临床干预提供新的靶点和契机。Hotchkiss等［9］发现，通过脓毒症鼠过度表达Bcl-2基因，几乎可以完全阻止脓毒症所诱导的淋巴细胞凋亡。由此预测，调控Bcl-2相关的细胞凋亡过程，有可能改变脓毒症的免疫功能状态。 　　miRNAs是一类长度约22个核苷酸大小的内源性非编码RNA，它可通过识别靶基因mRNA的3’端非编码区（3’UTR），并与之结合阻止翻译或导致mRNA降解，从而抑制靶基因的表达［10-12］。Taganov等［13］用LPS刺激人单核细胞，miRNA芯片检测发现miR-146a、miR-155和miR-132的表达量增高，并证实LPS是通过TLR4的信号通路调控miRNAs的表达变化，其中miR-146a是以IRAK-1（IL-1受体相关激酶1）和TRAF6（TNF受体相关因子6为靶点，通过调节NF-κB来实现对TLR4/IL-4信号途径的负调控；曾振国等［14］研究应用LPS刺激NR8383肺泡巨噬细胞，miR-146a的表达上调，且随着刺激时间的延长，miR-146a的表达有增高的趋势。Pauley等［15］则通过转染miR-146a拟似物进入THP-1细胞，证实可以下调由LPS诱导的细胞因子表达，但关于miRNA对脓毒症的发病过程的报道目前仍较缺乏。 　　Xiong等［6］的研究结果表明，miR-29参与肿瘤细胞的凋亡调控，在肝癌细胞中miR-29的表达水平降低，在肝癌细胞中过表达miR-29能促进肝癌细胞的凋亡，但miR-29能否调控免疫细胞凋亡，目前国内外未见报道。本实验先用LPS 诱导THP-1 细胞，能使细胞的凋亡水平增高，证实LPS能诱导THP-1 细胞凋亡。再通过细胞转染的方法，将合成的miR-29a的拟似物导入THP-1细胞中，达到过表达miR-29a的目的，结果显示THP-1的细胞凋亡水平增高，这与LPS诱导THP-1 细胞凋亡的效应类似；接着，THP-1 细胞先转染miR-29a 的inhibitor，阻断细胞内miR-29a的作用，再用LPS诱导细胞，结果细胞凋亡水平较单纯用LPS刺激降低，提示阻遏miR-29a的作用，能抑制LPS诱导的细胞凋亡。本研究应用生物信息学软件Targetscan，寻找miR-29a直接作用的靶点，发现抗凋亡基因Bcl-2 和Mcl-1的mRNA序列中，它们的3’UTR非编码区有能与miR-29a结合的种子序列，因此，在THP-1中过表达miR-29a后，用real time RT-PCR方法检测Bcl-2 和Mcl-1 mRNA表达水平，结果发现它们的表达水平下降，提示过表达miR-29a能抑制Bcl-2 和Mcl-1表达；Luciferase 实验证实，miR-29a可特异性抑制带有 Bcl-2 和Mcl-1 3’UTR上野生型识别元件的报告基因表达（P<0.05），证实了Bcl-2 和Mcl-1是miR-29a的靶基因，这与Xiong等［6］的研究结果一致。 　　综上所述，miRNA参与脓毒症细胞免疫功能调节过程，本研究结果显示miR-29a能通过靶向于两个抗凋亡基因Bcl-2和Mcl-1调控THP-1细胞凋亡水平，提示miR-29a在调控免疫细胞的凋亡过程中具有重要的作用，借助miR-29a进行免疫细胞凋亡水平调控，有望能改善脓毒症免疫细胞功能状态。 　　参考文献 　　［1］ Cohen J. 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Immunol Cell Biol， 2010， 88（2）：205-212. 　　（收稿日期：） 　　（本文编辑：邵菊芳） 　　DOI：10.3760/cma.j.issn.13.01.009 　　基金项目：广东省自然科学基金（00018）；广州市医药卫生科技项目（020） 　　作者单位：510260 广州，广州医学院第二附属医院重症医学科 　　P40-P45
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