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簡介：ORIGINALARTICLEALIMENTARYTRACTDOWNREGULATIONOFMIR141INGASTRICCANCERANDITSINVOLVEMENTINCELLGROWTHYINGDU?YANJUNXU?LINGDING?HAOMIYAO?HONGYU?TIANHUAZHOU?JIANMINSIRECEIVED18AUGUST2008/ACCEPTED28JANUARY2009/PUBLISHEDONLINE11APRIL2009?SPRINGER2009ABSTRACTPURPOSEHUMANMICRORNA141MIR141,AMEMBEROFTHEMIR200FAMILY,HASBEENREPORTEDTOBEASSOCIATEDWITHVARIOUSHUMANMALIGNANCIESHOWEVER,ITREMAINSUNKNOWNWHETHERMIR141ISINVOLVEDINTHEPATHOGENESISOFGASTRICCANCERTHEREFORE,WEEXAMINEDTHEEXPRESSIONOFMIR141INGASTRICCANCERTISSUESANDTHEEFFECTOFMIR141OVEREXPRESSIONONCANCERCELLPROLIFERATIONMETHODSTHEEXPRESSIONLEVELOFMIR141IN35PAIRMATCHEDGASTRICNEOPLASTICANDADJACENTNONNEOPLASTICTISSUES,ANDIN5GASTRICCANCERCELLLINESWEREEXAMINEDBYQUANTITATIVEREALTIMEPCRTHEGROWTHOFMGC803CELLSTRANSFECTEDWITHMIRNAPRECURSORWASEXAMINEDBYMTT34,5DIMETHYLTHIAZOL2YL2,5DIPHENYLTETRAZOLIUMBROMIDEASSAYRESULTSMIR141WASSIGNIFICANTLYDOWNREGULATEDIN8028/35OFPRIMARYGASTRICCANCERTISSUESCOMPAREDWITHPAIRMATCHEDADJACENTNONTUMORTISSUESP\001THEEXPRESSIONOFMIR141WASALSOFOUNDTOBESUBSTANTIALLYREDUCEDINSEVERALHUMANGASTRICCANCERCELLLINESSUCHASMGC803,HGC27,SGC7901ANDBGC823CELLSOVEREXPRESSIONOFMIR141WITHITSPRECURSORSSIGNIFICANTLYINHIBITEDTHEPROLIFERATIONOFGASTRICCANCERCELLSCONCLUSIONSTHESERESULTSSUGGESTTHATMIR141MAYBEINVOLVEDINTHEDEVELOPMENTOFGASTRICCANCERTHROUGHITSINHIBITORYEFFECTONCELLPROLIFERATIONKEYWORDSMIR141?GASTRICCANCER?MGC803CELL?CELLPROLIFERATIONINTRODUCTIONMICRORNASMIRNAS,ANEWCLASSOFENDOGENOUS,NONCODINGANDSINGLESTRANDEDRNAS,WERERECENTLYDISCOVEREDINBOTHANIMALSANDPLANTSTHEYTRIGGERTRANSLATIONALREPRESSIONAND/ORMRNADEGRADATIONMOSTLYTHROUGHCOMPLEMENTARYBINDINGTOTHE30UNTRANSLATEDREGIONSOFTARGETMRNASSTUDIESHAVESHOWNTHATMIRNASCANREGULATEAWIDEARRAYOFBIOLOGICALPROCESSESSUCHASCELLPROLIFERATION,DIFFERENTIATION,ANDAPOPTOSIS1ACCUMULATINGEVIDENCESUGGESTSTHATALTERATIONSOFMIRNASEXPRESSIONMAYPLAYVARIOUSROLESINTHEPATHOGENESISOFMANYHUMANCANCERS2,3SOMEMIRNASHASBEENSHOWNTOPOSSESSONCOGENICORTUMORSUPPRESSORACTIVITY4HIGHTHROUGHPUTTECHNIQUESHAVEBEENUSEDTOSCREENMIRNASDIFFERENTIALLYEXPRESSEDBETWEENHUMANNONMALIGNANTANDMALIGNANTSAMPLES,ANDANUMBEROFMIRNASDEREGULATEDINNUMEROUSHUMANTUMORSWEREFOUND,INCLUDINGLUNG,BREAST,LIVER,ESOPHAGEALANDPROSTATECANCERS5–9AMONGTHESEMIRNAS,MIR141,AMEMBEROFTHEMIR200FAMILY,ISOVEREXPRESSEDINOVARIANANDCOLORECTALCANCERS10,11ANDDOWNREGULATEDINPROSTATE,HEPATOCELLULAR,ANDRENALCELLCARCINOMA5,9,12,RAISINGACONTROVERSIALISSUEABOUTTHEROLEOFMIR141INCANCERPROGRESSIONELECTRONICSUPPLEMENTARYMATERIALTHEONLINEVERSIONOFTHISARTICLEDOI101007/S0053500900377CONTAINSSUPPLEMENTARYMATERIAL,WHICHISAVAILABLETOAUTHORIZEDUSERSYDU?HYU?JSIP\001SUBSTANTIALLYREDUCEDEXPRESSIONSOFMIR141WEREFOUNDINBOTHINTESTINALTYPE17FOLDN23FIG1BP\005ANDDIFFUSETYPEGASTRICCANCERS30FOLDN9FIG1CP\001THEEXPRESSIONOFMIR141INCELLLINESDERIVEDFROMGASTRICCANCERTOCONFIRMTHEASSOCIATIONBETWEENMIR141EXPRESSIONANDGASTRICCANCER,WEDETECTEDMIR141EXPRESSIONINFIG1THEEXPRESSIONOFMIR141INGASTRICCANCERQUANTIFICATIONOFMIR141WASMEASUREDBYTAQMANREALTIMEPCRALLDATAOFFOLDCHANGEWERETRANSFORMEDTOLOG2VALUESARELATIVEEXPRESSIONOFMIR141IN35PRIMARYGASTRICCANCERTISSUESCOMPAREDWITHTHEIRPAIRMATCHEDADJACENTNONTUMORTISSUESBRELATIVEEXPRESSIONOFMIR141IN23TISSUESWITHINTESTINALTYPEADENOCARCINOMARELATIVETOTHEIRPAIRMATCHEDADJACENTNONMALIGNANTTISSUESCRELATIVEEXPRESSIONOFMIR141IN9DIFFUSETYPEADENOCARCINOMATISSUESCOMPAREDWITHTHEIRPAIRMATCHEDADJACENTNONTUMORTISSUESTHEPATHOLOGICALFEATURESOFREPRESENTATIVEINTESTINALTYPEANDDIFFUSETYPEGASTRICCANCERWERESHOWNWITHHESTAININGSCALEBARS200LM558JGASTROENTEROL200944556–561123

簡介：ULTRASENSITIVEELECTRICALBIOSENSINGOFPROTEINSANDDNACARBONNANOTUBEDERIVEDAMPLIFICATIONOFTHERECOGNITIONANDTRANSDUCTIONEVENTSJOSEPHWANG,GUODONGLIU,ANDMRASULJAN?DEPARTMENTOFCHEMISTRYANDBIOCHEMISTRY,NEWMEXICOSTATEUNIVERSITY,LASCRUCES,NEWMEXICO88003RECEIVEDDECEMBER15,2003EMAILJOEWANGNMSUEDUTHEDETECTIONOFDNAANDPROTEINSISOFCENTRALIMPORTANCETOTHEDIAGNOSISANDTREATMENTOFGENETICDISEASES,TOTHEDETECTIONOFINFECTIOUSAGENTS,DRUGDISCOVERY,ORWARNINGAGAINSTBIOWARFAREAGENTS14SUCHBIODETECTIONCOMMONLYRELIESONHYBRIDIZATIONORANTIGENANTIBODYAGABINTERACTIONS,ANDREQUIRESPROPERATTENTIONTOTHEACHIEVEMENTOFULTRASENSITIVEMEASUREMENTSELECTROCHEMICALTRANSDUCERSAREVERYATTRACTIVEFORSUCHBIOASSAYS,OWINGTOTHEIRHIGHSENSITIVITY,INHERENTSIMPLICITYANDMINIATURIZATION,ANDLOWCOSTANDPOWERREQUIREMENTSTHEUSEOFENZYMELABELSTOGENERATEELECTRICALSIGNALSHASBEENEXTREMELYUSEFULFORULTRASENSITIVEELECTROCHEMICALBIOAFFINITYASSAYSOFPROTEINSANDDNAHELLER’SGROUP5,6DEMONSTRATEDTHATAHIGHLYSENSITIVEAMPEROMETRICMONITORINGOFDNAHYBRIDIZATIONDOWNTO5ZMOLCOULDBEACHIEVEDINCONNECTIONWITHAHORSERADISHPEROXIDASEHRPLABELEDTARGETANDANELECTRONCONDUCTINGREDOXPOLYMERHRPLABELHASBEENCOMBINEDBYWILLNER’SGROUP7,8WITHABIOCATALYTICPRECIPITATIVEACCUMULATIONOFTHEENZYMEGENERATINGPRODUCTTOACHIEVEMULTIPLEAMPLIFICATIONSANDVERYLOW25AMOLDETECTIONLIMITSEFFORTSTOAMPLIFYENZYMELINKEDELECTRICALPROTEINASSAYSINCLUDEDDUALENZYMESUBSTRATERECYCLING9ORIONEXCHANGEACCUMULATIONOFTHEPRODUCT10YET,AMPLIFIEDTRANSDUCTIONOFBIOLOGICALRECOGNITIONEVENTSREMAINSAMAJORCHALLENGETOELECTRICALBIOASSAYSNEWSCHEMESBASEDONCOUPLINGTHEBIOCATALYTICAMPLIFICATIONOFENZYMETAGSWITHADDITIONALAMPLIFICATIONUNITSANDPROCESSESAREHIGHLYDESIREDFORMEETINGTHEHIGHSENSITIVITYDEMANDSOFELECTROCHEMICALDETECTIONOFPROTEINSANDNUCLEICACIDSHEREWEDEMONSTRATETHEUSEOFCARBONNANOTUBESCNTSFORDRAMATICALLYAMPLIFYINGENZYMEBASEDBIOAFFINITYELECTRICALSENSINGOFPROTEINSANDDNATHEUNIQUEELECTRONIC,CHEMICAL,ANDMECHANICALPROPERTIESOFCNTSMAKETHEMEXTREMELYATTRACTIVEFORELECTROCHEMICALSENSORS11,12MOSTCNTSENSINGWORKHASFOCUSEDONTHEABILITYOFSURFACECONFINEDCNTSTOPROMOTEELECTRONTRANSFERREACTIONSINVOLVEDINBIOCATALYTICDEVICES13,14INOURNEWBIOAFFINITYASSAYSFIGURE1,CNTSPLAYADUALAMPLIFICATIONROLEINBOTHTHERECOGNITIONANDTRANSDUCTIONEVENTS,NAMELYASCARRIERSFORNUMEROUSENZYMETAGSANDFORACCUMULATINGTHEPRODUCTOFTHEENZYMATICREACTIONTHESENOVELSUPPORTANDPRECONCENTRATIONFUNCTIONSOFCNTSREFLECTTHEIRLARGESPECIFICSURFACEAREA15ANDAREILLUSTRATEDUSINGTHEALKALINEPHOSPHATASEALPENZYMETRACERSUCHCOUPLINGOFSEVERALCNTDERIVEDAMPLIFICATIONPROCESSESLEADSTOTHELOWESTDETECTIONLIMITREPORTEDTHUSFARFORELECTRICALDNADETECTIONTHENEWCNTBASEDAMPLIFIEDBIOELECTRONICPROTOCOLFIGURE1INVOLVESTHESANDWICHHYBRIDIZATIONAORANTIGENANTIBODYBBINDINGALONGWITHMAGNETICSEPARATIONOFTHEANALYTELINKEDMAGNETICBEAD/CNTASSEMBLYA,FOLLOWEDBYENZYMATICAMPLIFICATIONB,ANDCHRONOPOTENTIOMETRICSTRIPPINGDETECTIONOFTHEPRODUCTATTHECNTMODIFIEDELECTRODECOURTEMOBSERVATIONSEG,FIGURE2INDICATETHATTHEHYBRIDIZATIONEVENTLEADSTOCROSSLINKINGOFTHEALPLOADEDCNTSANDTHEMAGNETICBEADSWITHTHEDNADUPLEXACTINGAS“GLUE”TOOURKNOWLEDGE,THISISTHEFIRSTEXAMPLEOFUSINGDNAFORLINKINGPARTICLESTOCNTSNOSUCHAGGREGATIONWASOBSERVEDINTHEPRESENCEOFNONCOMPLEMENTARYOLIGONUCLEOTIDESBAPPARENTLY,WITHOUTTHERECOGNITIONEVENT,THEALPTAGGEDCNTSAREREMOVEDBYTHEMAGNETICSEPARATION,LEAVINGTHEMAGNETICBEADSBEHINDALPWASIMMOBILIZEDONCNTSUSINGA1ETHYL33DIMETHYLAMINOPROPYLCARBODIIMIDELINKERSEEFIGURE1INSUPPORTINGINFORMATIONACOVERAGEOFAROUND9600ENZYMEMOLECULESPERACNTIE,BINDINGEVENTWASESTIMATEDFROMASEPARATEELECTROCHEMICALEXPERIMENTCOMPARINGTHERNAPHTHOLRESPONSEOFKNOWNAMOUNTSOFALPLOADEDCNTSANDALPASSUMINGSIMILARACTIVITIESFORTHEFREEANDBOUNDALPTHEDRAMATICSIGNALENHANCEMENTASSOCIATEDWITHTHECNTBASEDDUALAMPLIFICATIONROUTEISDEMONSTRATEDINFIGURE3FORDNAHYBRIDIZATIONAANDAGABBBIOASSAYSTHECONVENTIONALPROTOCOLS,BASEDONTHESINGLEENZYMETAGANDAGLASSYCARBONTRANSDUCER,ARENOTRESPONDINGTOEITHER10PGML1DNATARGETA,AOR80PGML1IGGB,ATHEFIRSTAMPLIFICATIONSTEPBASEDONTHEALPLOADEDCNTSBOFFERSCONVENIENTDETECTIONOFTHESELOWANALYTECONCENTRATIONSTHESINGLEALPPROTOCOLSDISPLAYEDA?PERMANENTADDRESSDEPARTMENTOFCHEMISTRY,UNIVERSITYOFPESHAWAR,PAKISTANFIGURE1SCHEMATICREPRESENTATIONOFTHEANALYTICALPROTOCOLACAPTUREOFTHEALPLOADEDCNTTAGSTOTHESTREPTAVIDINMODIFIEDMAGNETICBEADSBYASANDWICHDNAHYBRIDIZATIONAORABAGABINTERACTIONBBENZYMATICREACTIONCELECTROCHEMICALDETECTIONOFTHEPRODUCTOFTHEENZYMATICREACTIONATTHECNTMODIFIEDGLASSYCARBONELECTRODEMB,MAGNETICBEADSP,DNAPROBE1T,DNATARGETP2,DNAPROBE2AB1,FIRSTANTIBODYAG,ANTIGENAB2,SECONDARYANTIBODYSANDP,SUBSTRATEANDPRODUCT,RESPECTIVELY,OFTHEENZYMATICREACTIONGC,GLASSYCARBONELECTRODECNT,CARBONNANOTUBELAYERFIGURE2TEMIMAGESOFTHEMAGNETICBEADSDNACNTASSEMBLYPRODUCEDFOLLOWINGA20MINHYBRIDIZATIONWITHTHE10AAND0BPGML1TARGETSAMPLETHEMICROGRAPHSWERETAKENWITHAHITACHIH7000INSTRUMENTOPERATEDAT75KVPUBLISHEDONWEB02/18/200430109JAMCHEMSOC2004,126,30103011101021/JA031723WCCC2750?2004AMERICANCHEMICALSOCIETYLOWERSIGNALFORASIGNIFICANTLY1000FOLDHIGHERTARGETCONCENTRATIONNOTSHOWNTHENEARLY104IMPROVEMENTINTHESENSITIVITYISINGOODAGREEMENTWITHTHEESTIMATEDALPLOADINGPERCNTONLY～50FOLDSENSITIVITYENHANCEMENTWASOBSERVEDBYUSINGASTREPTAVIDINCOATEDPOLYSTYRENECARRIERBEADINSTEADOFTHECNTSUPPORTFURTHERENHANCEMENTSOFTHEDNAANDPROTEINSIGNALSBY～30FOLDAREOBSERVEDINTHESECONDAMPLIFICATIONPATH,EMPLOYINGTHECNTMODIFIEDTRANSDUCERCTHELATTERREFLECTTHESTRONGADSORPTIVEACCUMULATIONOFTHELIBERATEDRNAPHTHOLONTHECNTLAYERTHEPRECONCENTRATIONFEATUREOFTHECNTLAYERWASINDICATEDFROMTHEUSEOFDIFFERENTACCUMULATIONTIMESTHATLEDTOASHARPINCREASEINTHERNAPHTHOLSIGNALCOMPAREDTOTHETIMEINDEPENDENTSIGNALOBSERVEDATTHEBAREELECTRODESEEFIGURE2INSUPPORTINGINFORMATIONFIGURE4ADISPLAYSTYPICALCHRONOPOTENTIOGRAMSFOREXTREMELYLOWTARGETDNACONCENTRATIONS001100PGML1AEWELLDEFINEDRNAPHTHOLSIGNALSAREOBSERVEDFORTHESELOWDNACONCENTRATIONSINCONNECTIONWITH20MINHYBRIDIZATIONTHERESULTINGPLOTOFRESPONSEVSLOGTARGETSHOWNASINSETISLINEARANDSUITABLEFORQUANTITATIVEWORKTHEFAVORABLERESPONSEOFTHE5FGML1DNATARGETBINDICATESAREMARKABLYLOWDETECTIONLIMITOFAROUND1FGML154AM,IE,820COPIESOR13ZMOLINTHE25ΜLSAMPLESUCHALOWDETECTIONLIMITCOMPARESFAVORABLYWITHTHELOWESTVALUESOF5ZMOL3000COPIESAND25AMOLREPORTEDFORELECTRICALDNADETECTION6,8SIMILARLY,IGGWASDETERMINEDWITHADETECTIONLIMITOF500FGML1160ZMOLIN25ΜLSAMPLESANDEXHIBITSAWELLDEFINEDLOGARITHMICCONCENTRATIONDEPENDENCETHESMALLERSIGNALOBSERVEDINACONTROLEXPERIMENTFORAHUGE～106EXCESSOFANONCOMPLEMENTARYOLIGONUCLEOTIDEFIGURE4,CVSBREFLECTSTHEHIGHSELECTIVITYASSOCIATEDWITHTHEEFFECTIVEMAGNETICSEPARATIONTHEAMPLIFIEDELECTRICALSIGNALISCOUPLEDTOAGOODREPRODUCIBILITYTWOSERIESOFSIXREPETITIVEMEASUREMENTSOF1PGML1DNATARGETOR08NGML1IGGYIELDEDREPRODUCIBLESIGNALSWITHRELATIVESTANDARDDEVIATIONSOF56AND89,RESPECTIVELYINCONCLUSION,WEHAVEDEMONSTRATEDACNTBASEDDUALAMPLIFICATIONROUTEFORULTRASENSITIVEELECTRICALBIOASSAYSOFPROTEINSANDDNATHEUSEOFCNTAMPLIFIERSLOADEDWITHNUMEROUSALPTAGSHASBEENCOMBINEDWITHTHEPRECONCENTRATIONFEATUREOFCNTTRANSDUCERSTOYIELDADRAMATICENHANCEMENTOFTHESENSITIVITYSUCHCOUPLINGOFSEVERALCNTDERIVEDAMPLIFICATIONPROCESSESRESULTSINHIGHLYSENSITIVEDETECTIONOFPROTEINSANDDNAANDHENCEINDICATESGREATPROMISEFORPCRFREEDNAASSAYSFURTHERIMPROVEMENTSINTHESENSITIVITYAREEXPECTEDEITHERTHROUGHREDUCINGTHEELECTRODESIZEANDSAMPLEVOLUME6ORBYSUBSTRATERECYCLING9THENEWCNTDERIVEDAMPLIFICATIONBIOASSAYSAREEXPECTEDTOOPENNEWOPPORTUNITIESFORMEDICALDIAGNOSTICSANDPROTEINANALYSISTHEFINDINGTHATDNAHYBRIDIZATIONCANBEUSEDFORLINKINGCNTSTOPARTICLESHOLDSPROMISEFORASSEMBLINGCONTROLLABLENANOSCALESYSTEMSACKNOWLEDGMENTFINANCIALSUPPORTFROMTHENATIONALSCIENCEFOUNDATIONGRANTCHE0209707ANDNATIONALINSTITUTESOFHEALTHAWARDR01A105604701ISGRATEFULLYACKNOWLEDGEDSUPPORTINGINFORMATIONAVAILABLERELATEDEXPERIMENTALCONDITIONSINSTRUMENTATION,REAGENTS,SEQUENCES,ANDPROCEDURESALONGWITHADDITIONALDATAPDFTHISMATERIALISAVAILABLEFREEOFCHARGEVIATHEINTERNETATHTTP//PUBSACSORGREFERENCES1PALECEK,EFOJTA,MANALCHEM2001,73,75A2DRUMMOND,TGHILL,MGBARTON,JKNATBIOTECHNOL2003,21,11923WANG,JCHEMEURJ1999,5,16814GOODING,JJELECTROANALYSIS2002,14,11495CARUANA,DJHELLER,AJAMCHEMSOC1999,121,7696ZHANG,YKIM,HHELLER,AANALCHEM2003,75,32677PATOLSKY,FKATZ,EBARDEA,AWILLNER,ILANGMUIR1996,12,37038PATOLSKY,FLITCHENSTEIN,AWILLNER,IANGEWCHEM,INTED2000,39,9409BAUER,CEREMENKO,AEHRENTREICHFOSTER,EBIER,FMAKOWER,AHALSALL,HBHEINEMAN,WRSCHELLER,FWANALCHEM1996,68,245310LIMOGES,BDEGRAND,CANALCHEM1996,68,414111BAUGHMAN,RHZAKHIDOV,ADEHEER,WASCIENCE2002,297,78712ZHAO,QGAN,ZZHUANG,QELECTROANALYSIS2002,14,160913WANG,JMUSAMEH,MLIN,YJAMCHEMSOC2003,125,240814RUBIANES,MDRIVAS,GAELECTROCHEMCOMMUN2003,5,68915PEIGNEY,ALAURENT,CFLAHAUT,EBASCA,RROUSSET,ACARBON2001,39,507JA031723WFIGURE3CHRONOPOTENTIOMETRICSIGNALSFOR10PGML1TARGETOLIGONUCLEOTIDEAAND80PGML1IGGBUSINGTHEGLASSYCARBONGCTRANSDUCERANDAASINGLEALPTAGANDBCNTLOADEDWITHMULTIPLEALPTAGSCSAMEASBBUTUSINGTHECNTMODIFIEDGCELECTRODEAMOUNTOFMAGNETICBEADS,50ΜGSANDWICHASSAYWITH20AND30MINFOREACHHYBRIDIZATIONEVENTANDAG/ABASSOCIATION,RESPECTIVELYSAMPLEVOLUME,50ΜLDETECTION,ADDITIONOF50ΜLRNAPHTHYLPHOSPHATE50MMSOLUTIONWITHA20MINENZYMATICREACTIONMEASUREMENTSOFTHERNAPHTHOLPRODUCTWEREPERFORMEDATTHEBAREORMODIFIEDGCELECTRODES,USINGA2MINACCUMULATIONAT02VINASTIRREDPHOSPHATEBUFFERSOLUTION005M,PH741ML,FOLLOWEDBYA10SRESTPERIODWITHOUTSTIRRINGANDAPPLICATIONOFANANODICCURRENTOF50ΜASEESUPPORTINGINFORMATIONFORTHECONCENTRATIONSOFTHEOLIGONUCLEOTIDEPROBESANDANTIBODY,ANDSEQUENCEOFOLIGONUCLEOTIDEPROBES,LEVELSANDPREPARATIONOFTHEALPDNACNTANDALPSTREPTAVIDINCNTCONJUGATESFIGURE4CHRONOPOTENTIOMETRICSIGNALSFORINCREASINGLEVELSOFTHEDNATARGETA001,B01,C1,D50,E100PGML1ALSOSHOWNINSETISTHERESULTINGCALIBRATIONPLOTA,ANDTHERESPONSEFOR5FGML1TARGETDNABAND10NGML1NONCOMPLEMENTARYNCOLIGONUCLEOTIDECSAMPLEVOLUME,25ΜLBAND50ΜLCOTHERCONDITIONS,ASINFIGURE3A,CBASEDONPROTOCOLOFFIGURE1AACCOMMUNICATIONSJAMCHEMSOC9VOL126,NO10,20043011

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