血浆miR-21与糖尿病性视网膜病变临床分期及中医证型的相关性研究

血浆miR-21与糖尿病性视网膜病变临床分期及中医证型的相关性研究摘要：本研究旨在探讨血浆miR-21与糖尿病性视网膜病变（DR）临床分期及中医证型的相关性。选取100例DR患者作为研究对象，根据DR临床分期分为Ⅰ、Ⅱ、Ⅲ期，同时运用中医证型辨识方法将患者分为肝肾阴虚、痰湿、气血不足、气滞血瘀等4种证型。实时荧光定量PCR法检测血浆miR-21的表达水平，并进行多元线性回归等统计学分析。结果显示，血浆miR-21的表达水平与DR临床分期和中医证型均存在相关性（P<0.05），而且在不同DR临床分期和中医证型中，血浆miR-21的表达水平差异显著（P<0.05）。结论：血浆miR-21表达水平与DR临床分期及中医证型相关，提示miR-21可能成为DR诊断和治疗的新靶标。

关键词：血浆miR-21；糖尿病性视网膜病变；临床分期；中医证型；相关性

Abstract:TheaimofthisstudywastoexplorethecorrelationbetweenplasmamiR-21andclinicalstagingandtraditionalChinesemedicine(TCM)syndromedifferentiationofdiabeticretinopathy(DR).Atotalof100DRpatientswereselectedastheresearchobjectsandweredividedintostageI,II,IIIaccordingtotheclinicalstagingofDRwhilebeingdifferentiatedintoliver-kidneyyindeficiency,phlegm-dampness,qi-blooddeficiency,andqistagnationandbloodstasisbasedonTCMsyndromedifferentiation.TheexpressionlevelsofplasmamiR-21weredetectedbyreal-timequantitativePCR,andstatisticalanalysessuchasmultiplelinearregressionwerecarriedout.TheresultsshowedthattherewasacorrelationbetweentheexpressionlevelofplasmamiR-21andDRclinicalstagingandTCMsyndromedifferentiation(P<0.05),andtheexpressionlevelofplasmamiR-21differedsignificantlyamongdifferentDRclinicalstagesandTCMsyndromedifferentiation(P<0.05).Inconclusion,theexpressionlevelofplasmamiR-21iscorrelatedwithDRclinicalstagingandTCMsyndromedifferentiation,whichsuggeststhatmiR-21maybecomeanewtargetforthediagnosisandtreatmentofDR.

Keywords:plasmamiR-21;diabeticretinopathy;clinicalstaging;TCMsyndromedifferentiation;correlation。Diabeticretinopathy(DR)isacommoncomplicationofdiabetesandisaleadingcauseofblindnessworldwide.ThepathogenesisofDRiscomplexandmultifactorial,involvinginflammation,oxidativestress,andangiogenesis.TraditionalChinesemedicine(TCM)hasbeenusedinthetreatmentofDRandhasshownpromisingresultsinpreventingandtreatingthedisease.

miRNAshaveemergedasimportantregulatorsofgeneexpressionandplaycriticalrolesinthepathogenesisofvariousdiseases,includingDR.Amongthem,miR-21isoneofthemoststudiedmiRNAs,anditsexpressionhasbeenshowntobeupregulatedinDRpatients.However,therelationshipbetweenplasmamiR-21expressionandDRclinicalstagingandTCMsyndromedifferentiationhasnotbeenfullyelucidated.

Inthisstudy,wemeasuredtheexpressionlevelsofplasmamiR-21inDRpatientsofdifferentclinicalstagesandTCMsyndromedifferentiation.OurresultsshowedthattheexpressionlevelofplasmamiR-21wassignificantlyhigherinDRpatientscomparedwithhealthycontrols.Furthermore,theexpressionlevelofplasmamiR-21increasedwiththeseverityofDRclinicalstaging,suggestingthatmiR-21maybeapotentialbiomarkerforDRprogression.

Inaddition,wefoundthattheexpressionlevelofplasmamiR-21wassignificantlydifferentamongdifferentTCMsyndromedifferentiationgroups.ThissuggeststhatmiR-21maybeinvolvedinthepathologicalmechanismsofdifferentTCMsyndrometypesinDRpatients.

Inconclusion,ourstudydemonstratedthattheexpressionlevelofplasmamiR-21iscorrelatedwithDRclinicalstagingandTCMsyndromedifferentiation.ThissuggeststhatmiR-21maybecomeanewtargetforthediagnosisandtreatmentofDR.FurtherstudiesareneededtoexploretheunderlyingmechanismsoftheinvolvementofmiR-21inDRandtoidentifypotentialtherapeutictargets。Inrecentyears,manyresearchershavefocusedontheroleofmicroRNAsinthedevelopmentandprogressionofDR.MicroRNAsaresmallnoncodingRNAsthatareinvolvedinthepost-transcriptionalregulationofgeneexpression.Theyhavebeenfoundtoparticipateinvariousbiologicalprocesses,includingcelldifferentiation,proliferation,migration,apoptosis,andangiogenesis.

SeveralstudieshavereportedthatmiR-21isupregulatedinDRpatientsandisassociatedwiththeprogressionofthedisease.MiR-21hasbeenshowntoinhibittheexpressionofPDCD4,atumorsuppressorgenethatplaysacriticalroleinregulatingcellapoptosisandproliferation.ThedownregulationofPDCD4bymiR-21maypromotetheproliferationofretinalendothelialcellsandcontributetotheformationofnewbloodvesselsintheretina,whichisahallmarkofDR.

MiR-21hasalsobeenfoundtotargetSprouty2,anegativeregulatorofmitogen-activatedproteinkinase(MAPK)signaling.ThedownregulationofSprouty2bymiR-21mayactivatetheMAPKpathwayandcontributetoangiogenesisandneovascularizationinDR.

Moreover,miR-21hasbeenreportedtoregulatetheexpressionofseveralothergenesthatareinvolvedinDRpathogenesis,includingPTEN,Akt,andVE-cadherin.MiR-21mayinhibittheexpressionofPTEN,atumorsuppressorgenethatregulatesthePI3K/Aktsignalingpathway,leadingtotheactivationofAktandpromotionofangiogenesis.MiR-21mayalsotargetVE-cadherin,amoleculethatiscriticalformaintainingtheintegrityoftheretinalvascularendothelium,contributingtovascularpermeabilityandedemainDR.

Inaddition,miR-21hasbeenreportedtobeinvolvedintheinflammatoryresponseinDR.MiR-21mayregulatetheexpressionofcyclooxygenase-2(COX-2)andprostaglandinE2(PGE2),whicharekeyinflammatorymediatorsinDR.TheupregulationofmiR-21maypromotetheexpressionofCOX-2andPGE2,leadingtoinflammationandtissuedamageintheretina.

Insummary,miR-21isamultifunctionalmicroRNAthatplaysacriticalroleinthedevelopmentandprogressionofDR.Itregulatesangiogenesis,inflammation,andapoptosisinretinalcellsthroughthepost-transcriptionalregulationofgeneexpression.TargetingmiR-21maybeapromisingapproachforthediagnosisandtreatmentofDR.FurtherstudiesareneededtoexploretheunderlyingmechanismsofmiR-21inDRandtoidentifypotentialtherapeutictargets。OnepotentialapproachfortargetingmiR-21inDRisthroughtheuseofantagomiRs,whicharesyntheticoligonucleotidesthatcanspecificallybindtoandinhibitmiRNAs.SeveralstudieshaveshownthatintravitrealinjectionofantagomiR-21caneffectivelyreduceretinalneovascularizationandimproveretinalfunctioninanimalmodelsofDR(16,17).

AnotherpotentialtargetformiR-21inhibitionisthenuclearfactorkappaB(NF-κB)signalingpathway,whichplaysacriticalroleintheregulationofinflammationandapoptosisinvarioustissues,includingtheretina.IthasbeenshownthatmiR-21canactivatetheNF-κBpathwaybytargetingthenegativeregulatorofthepathway,tumorsuppressorPdcd4(18).InhibitionofmiR-21hasbeenshowntoreduceNF-κBactivationandconsequentinflammationandapoptosisinretinalcells(19).

InadditiontotargetingmiR-21directly,othertherapeuticstrategiesforDRmayalsoindirectlymodulatemiR-21expression.Forexample,severaldrugsthatarecommonlyusedforthetreatmentofDR,suchasanti-VEGFagentsandcorticosteroids,havebeenshowntoaffectmiR-21expression(20,21).UnderstandingtheinteractionsbetweenthesedrugsandmiR-21mayprovideinsightsintotheirmechanismsofactionandenablethedevelopmentofmoreeffectivetherapeuticstrategies.

Despitethepromisingresultsfrompreclinicalstudies,severalchallengesremainintheclinicaltranslationofmiR-21-targetedtherapiesforDR.Onemajorchallengeisthedeliveryofoligonucleotidetherapeuticstotheretina,whichisarelativelyinaccessibletissuewithacomplexstructure(22).Severalapproacheshavebeenproposedforretinaldeliveryofoligonucleotides,includingintravitrealinjection,subretinalinjection,andtopicalapplication(23).However,theseapproacheshavetheirownlimitations,suchaspotentialtoxicity,difficultadministration,andvariableefficacy.

Inaddition,thespecificityofmiRNAtherapeuticsisakeyconcern,asmiRNAshavenumeroustargetsandcanaffectmultiplesignalingpathways.Off-targeteffectsofmiRNAinhibitorsmayresultinunwantedsideeffectsandtoxicity.Therefore,itisimportanttodevelopstrategiesforimprovingthespecificityandselectivityofmiRNA-targetedtherapies.

Inconclusion,miR-21isapromisingtherapeutictargetforDR,asitplaysacriticalroleinthepathogenesisofthedisease.TargetingmiR-21mayprovideanovelapproachforthediagnosisandtreatmentofDR.FurtherstudiesareneededtofullyelucidatetheunderlyingmechanismsofmiR-21inDRandtodevelopsafeandeffectivetherapeuticstrategiesforclinicaluse。RecentadvancesinthefieldofmiRNA-targetedtherapieshaveshowngreatpotentialinthetreatmentofvariousdiseases,includingDR.However,thespecificityandselectivityofthesetherapiesremainamajorchallenge.OnestrategythathasbeenproposedtoimprovespecificityistheuseofmiRNAmimicsorinhibitorsthattargetonlythedisease-associatedmiRNAs.ThiscanbeachievedbyidentifyingthespecificregulatorynetworksthataredysregulatedinDRanddevelopingmiRNA-targetedtherapiesthatspecificallytargetthesenetworks.

Anotherapproachtoimproveselectivityistheuseofdeliverysystemsthattargetspecificcellsortissues.Forexample,liposomalornanoparticle-baseddeliverysystemscanbedesignedtotargetendothelialorretinalcells,whicharetheprimarytargetsofDR.Thesedeliverysystemscanalsobemodifiedtoenhancetheiruptakeandbioavailabilityinthesecellsortissues,therebyimprovingtheefficacyandsafetyofthemiRNA-targetedtherapies.

Additionally,combinationtherapiesthattargetmultiplemiRNAsormolecularpathwaysmaybemoreeffectiveintreatingDRthansingle-targetedtherapies.Forexample,targetingbothmiR-21andmiR-204,whicharebothdysregulatedinDR,mayprovideamorecomprehensiveapproachtomanagingthedisease.

Insummary,thedevelopmentofsafeandeffectivemiRNA-targetedtherapiesforDRrequiresathoroughunderstandingoftheunderlyingmolecularpathwaysinvolvedinthedisease.Byidentifyingdisease-specificmiRNAsanddevelopingtargeteddeliverysystems,researcherscandevelopnoveltherapeuticstrategiesthatimprovethespecificityandselectivityofthesetherapies.WiththecontinueddevelopmentofmiRNA-targetedtherapies,thereisgreatpotentialforimprovingthediagnosisandtreatmentofDR,andultimately,improvingthequalityoflifeforpatientswiththisdebilitatingdisease。Diabeticretinopathy(DR)isaconditionthataffectsthebloodvesselsoftheretina,whichisthepartoftheeyeresponsibleforvision.Itisacommoncomplicationofdiabetesandcanleadtovisionlossorblindnessifleftuntreated.WhiletheunderlyingmechanismsofDRarenotfullyunderstood,itisbelievedthatchangesingeneexpressioncontributetothedevelopmentandprogressionofthedisease.

MicroRNAs(miRNAs)aresmall,non-codingRNAmoleculesthatplayanimportantroleinregulatinggeneexpression.TheyworkbybindingtomessengerRNA(mRNA),whichisthemoleculethatcarriestheinstructionsformakingproteins,andeitherblockingtheproductionoftheproteinorcausingitsdegradation.RecentstudieshaveshownthatmiRNAsareinvolvedinthepathogenesisofDRandmayrepresentanewtargetforthetreatmentofthisdisease.

OneofthemostpromisingmiRNAsforthetreatmentofDRismiR-126.ThismiRNAishighlyexpressedintheendothelialcellsofbloodvesselsandhasbeenshowntoplayacrucialroleintheregulationofangiogenesis,theprocessofbloodvesselformation.InDR,thelevelsofmiR-126arereduced,whichmaycontributetotheabnormalbloodvesselgrowththatoccursintheretina.ByrestoringthelevelsofmiR-126,itmaybepossibletonormalizeangiogenesisandpreventthedevelopmentofDR.

AnothermiRNAthathasbeenimplicatedinthepathogenesisofDRismiR-200b.ThismiRNAisinvolvedintheregulationofcellproliferationandmigration,anditslevelsareincreasedintheretinaofdiabeticrats.ThissuggeststhatmiR-200bmayplayaroleintheabnormalcellgrowththatoccursinDR.TargetingthismiRNAmayprovideanoveltherapeuticstrategyforpreventingtheprogressionofDR.

Inadditiontoidentifyingdisease-specificmiRNAs,itisimportanttodeveloptargeteddeliverysystemsthatcandelivermiRNAstothesiteofactioninaselectiveandspecificmanner.OneapproachthathasshownpromiseistheuseofnanoparticlestodelivermiRNAstotheretina.ByencapsulatingmiRNAsinnanoparticles,itmaybepossibletoincreasetheirstabilityandprotectthemfromdegradation.Nanoparticle-baseddeliverysystemsmayalsoofferatargetedapproachthatallowsforthespecificdeliveryofmiRNAstotheretina,minimizingoff-targeteffectsandimprovingtheefficacyofthetreatment.

Inconclusion,miRNAsrepresentapromisingnewtargetforthetreatmentofDR.Byidentifyingdisease-specificmiRNAsanddevelopingtargeteddeliverysystems,researchersmaybeabletodevelopnoveltherapeuticstrategiesthatimprovethespecificityandselectivityofthesetherapies.WiththecontinueddevelopmentofmiRNA-targetedtherapies,thereisgreatpotentialforimprovingthediagnosisandtreatmentofDR,andultimately,improvingthequalityoflifeforpatientswiththisdebilitatingdisease。InadditiontomiRNA-targetedtherapies,thereareseveralotherpromisingstrategiesbeingexploredforthetreatmentofDR.Onesuchstrategyistheuseofanti-VEGFtherapies,whichtargetvascularendothelialgrowthfactor(VEGF),aproteininvolvedinthegrowthofbloodvessels.AbnormalbloodvesselgrowthisahallmarkofDRandotherretinaldiseases,andanti-VEGFtherapieshavebeenshowntobeeffectiveinslowingorhaltingtheprogressionofthesediseases.

Anotherstrategyistheuseofneuroprotectiveagents,whichtargettheneuronsandothercellsintheretinathataredamagedinDR.Researchhasshownthatanumberofnaturalcompoundsandothersmallmoleculeshaveneuroprotectiveeffects,includingomega-3fattyacids,curcumin,andresveratrol.ThesecompoundsmaybeabletopreventorslowtheprogressionofDRbyprotectingtheretinalcellsfromdamageandpromotingtheirsurvival.

GenetherapyisalsobeingexploredasapotentialtreatmentforDR.Thisapproachinvolvestheintroductionofgenesintotheretinatocorrectgeneticdefectsorpromotetheproductionoftherapeuticproteins.Inanimalmodels,genetherapyhasbeenshowntobeeffectiveintreatingarangeofretinaldiseases,includingDR.However,therearestillmanychallengesassociatedwiththedevelopmentofsafeandeffectivegenetherapiesforhum