循环的microRNA是早期诊断人类急性心肌梗死的潜在的新型生物标志物

CirculatingmicroRNA:anovelpotentialbiomarkerforearlydiagnosisofacutemyocardialinfarctioninhumansMicroRNA(miRNA)isreportedtobepresentinthebloodofhumansandhasbeenincreasinglysuggestedasabiomarkerfordiseases.Weaimtodeterminethepotentialofcardiac-specificmiRNAsincirculationtoserveasbiomarkersforacutemyocardialinfarction(AMI).Aims(1)Byverifyingtheirtissueexpressionpatternswithreal-timepolymerasechainreaction(PCR)analysis,muscle-enrichedmiRNAs(miR-1,miR-133a,andmiR-499)andcardiac-specificmiR-208awereselectedascandidatesforthisstudy.Methodsandresults(2)WithmiRNAmicroarrayandreal-timePCRanalyses,miR-1,miR-133a,andmiR-499werepresentwithverylowabundance,andmiR-208awasabsentintheplasmafromhealthypeople.IntheAMIrats,theplasmalevelsofthesemiRNAsweresignificantlyincreased.Especially,miR-208ainplasmawasundetectedat0h,butwassignificantlyincreasedtoadetectablelevelasearlyas1haftercoronaryarteryocclusion.Methodsandresults(3)Byreceiveroperatingcharacteristic(ROC)curveanalysis,amongthefourmiRNAsinvestigated,miR-208arevealedthehighersensitivityandspecificityfordiagnosingAMI.FurtherevaluationofthemiRNAlevelsinplasmafromAMIpatients(n=33)demonstratedthatallfourmiRNAlevelsweresubstantiallyhigherthanthosefromhealthypeople(n=30,P,0.01),patientswithnon-AMIcoronaryheartdisease(n=16,P,0.01),orpatientswithothercardiovasculardiseases(n=17,P,0.01).Notably,miR-208aremainedundetectableinnon-AMIpatients,butwaseasilydetectedin90.9%AMIpatientsandin100%AMIpatientswithin4hoftheonsetofsymptoms.ResultsFigure1TissueexpressionpatternsofmicroRNAsinratandhumantissues.(A)Real-timepolymerasechainreactionanalysisofmicroRNAexpressioninvarioustissuesfromrat.(B)Real-timepolymerasechainreactionanalysisofmicroRNAexpressioninvarioustissuesfromhuman.Sk.M,skeletalmuscle.ResultsFigure2microRNAsprofilinginhealthyhumanplasmaandcardiac-specificmicroRNAswereabsent.EqualoftotalRNAextractedfromhealthyhumanplasma(n=4)wasdetectedbymicroRNAmicroarrayanalysis.Y-axisrepresentstheaveragesignalintensityofthefourpeople.Thetop40microRNAswithdetectionsignalintensitywerepresent.ResultsTable1ConsistencyofplasmamicroRNAlevelsinhealthypeopledetectedbyreal-timepolymerasechainreactionandmicroarrayThedatawerepresentedasmean+SEM.Ct,thecyclenumberatgiventhresholdofreal-timePCR;UD,undetectable.ResultsFigure3IncreaseofmicroRNAsinplasmafromratswithacutemyocardialinfarction.(A)Theplasmawascollectedfromtheratsatdifferenttimesaftercoronaryarteryocclusion(n=6).TotalRNAwasisolated,reverse-transcribedandsubjectedtoreal-timepolymerasechainreactionanalysis.LevelsofmicroRNAswereexpressedasfoldincrease(logarithmicscale)relativetothosefromthecorrespondingratsat0h.(B)Theplasmawascollectedfromratsofnon-operation(non-op),sham-operation(sham-op),andligationgroupsat3haftercoronaryarteryocclusion(n=6,eachgroup).†Ctvaluesgreaterthan40weretreatedas40whennormalizingdata.*P0.05and**P,0.01.ResultsTable2ClinicalcharacteristicsofpatientswithandwithoutacutemyocardialinfarctionP1:comparisonbetweenpatientswithAMIandwithoutAMI.P2:comparisonamongpatientswithAMI,withCHD,andnon-CHD.ResultsFigure4Elevationofcardiac-specificmicroRNAsinplasmafromacutemyocardialinfarctionpatients.(A)Theplasmawascollectedfrompatientswithacutemyocardialinfarction(n=33),patientswithcoronaryheartdiseasebutwithoutacutemyocardialinfarction(coronaryheartdisease,n=16),patientswithothercardiovasculardisease(non-coronaryheartdisease,n=17)andhealthyvolunteers(healtn=30).TotalRNAwasisolated,reverse-transcribedandsubjectedtoreal-timepolymerasechainreactionanalysis.Darklinesrepresemeanormedianvalueswhereappropriate(**P0.01).Results(B)Theplasmawascollectedfromfivepatientswithacutemyocardialinfarctionafterreceiving2monthsofmedicaltreatment.ResultsFigure5EvaluationofplasmamicroRNAsforthediagnosisofacutemyocardialinfarction.(A)ReceiveroperatingcharacteristiccurvesweredrawnwiththedataofplasmamicroRNAsfrom66patientswithchestpain.AUC,theareaundercurve.(B)SensitivityandspecificityofplasmamicroRNAlevelsinthediagnosisofacutemyocardialinfarction.Thedashedlineindicatesa100%specificitythreshold.Filledcircle,acutemyocardialinfarctionpatients;invertedtriangle,non-acutemyocardialinfarctionpatients.Elevatedcardiac-specificmiR-208ainplasmamaybeanovelbiomarkerforearlydetectionofmyocardialinjuryinhumans.ConclusionDiscussionThepresentworkhasledustoidentifythatasetofmiRNAscanbeclinicallypracticablebiomarkersforAMIdiagnosis.IntheAMIanimalmodel,wefoundthattheplasmalevelsofmiRNAs,especiallythecardiac-specificmiR-208a,weresignificantlyincreasedafterAMI,suggestingthatcardiacmiRNAscouldreleaseintocirculatingbloodwhenheartinjuryoccurred.Microvesiclesaresmallvesiclesofendocyticoriginreleasedbynormalhealthyordamagedcellsandarereportedtobepresentintheperipheralblood.MoreevidencerevealsthatmiRNAsarepresentinmicrovesicles.Itremainstobeseenwhethercardiac-originmiRNAsreleasedfromdamagedmyocardiumareintheformofmicrovesicleswhentheyenterintocirculatingblood.