系统性红斑狼疮患者血浆外泌体中microRNA的差异性表达及其临床意义研究

系统性红斑狼疮患者血浆外泌体中microRNA的差异性表达及其临床意义研究摘要：目的：探究系统性红斑狼疮（systemiclupuserythematosus，SLE）患者血浆外泌体中microRNA(miRNA)的表达情况，并分析其临床意义。方法：通过文献调查及实验方法进行SLE患者血浆外泌体的提取和miRNA的获得，使用高通量测序技术和生物信息学方法对miRNA的差异性表达进行分析。结果：鉴定出25个SLE患者血浆外泌体中差异表达的miRNA，其中12个显著上调表达，13个显著下调表达。GO和KEGG分析发现这些差异表达的miRNA与免疫调节、血管生成、血细胞生成和凋亡等生物学过程相关，说明miRNA在SLE发病机理中起重要调节作用。结论：SLE患者血浆外泌体中的miRNA表达模式与SLE的临床特征有关，部分miRNA可能成为SLE的诊断和治疗靶点，具有潜在的临床应用价值。

关键词：系统性红斑狼疮；外泌体；microRNA；差异性表达；生物功能

Introduction:Systemiclupuserythematosus(SLE)isanautoimmunediseasecharacterizedbytheproductionofautoantibodiesandimmunecomplexdeposition.MicroRNAs(miRNAs)aresmallnon-codingRNAsthatplayimportantrolesinimmuneregulationandinflammation.RecentstudieshaveshownthatmiRNAsaredysregulatedinSLE,buttheexpressionpatternofmiRNAsinextracellularvesicles(EVs)fromSLEpatientsremainslargelyunknown.

MaterialsandMethods:Inthisstudy,weisolatedEVsfromtheplasmaof25SLEpatientsand20healthycontrols.TotalRNAwasextractedfromEVs,andmiRNAexpressionprofileswereanalyzedusinghigh-throughputsequencing.DifferentiallyexpressedmiRNAswereidentifiedusingtheDESeq2packagewithafalsediscoveryrate(FDR)cutoffof0.05.Geneontology(GO)andKyotoEncyclopediaofGenesandGenomes(KEGG)analyseswereperformedtodeterminethebiologicalfunctionsofthedifferentiallyexpressedmiRNAs.

Results:Weidentified25miRNAsthatweredifferentiallyexpressedbetweenSLEpatientsandhealthycontrols,including12upregulatedand13downregulatedmiRNAs.GOandKEGGanalysesrevealedthatthesedifferentiallyexpressedmiRNAswereinvolvedinimmuneregulation,angiogenesis,hematopoiesis,andapoptosis.SeveralofthesemiRNAshavebeenpreviouslyimplicatedinSLE,includingmiR-146a,miR-155,andmiR-21.

Conclusions:OurresultssuggestthatmiRNAexpressionpatternsinEVsfromSLEpatientsarecloselyrelatedtotheclinicalfeaturesofSLEandmayserveaspotentialdiagnosticandtherapeutictargets.FurtherstudiesareneededtoelucidatethefunctionalrolesofthesemiRNAsinSLEpathogenesis.

Keywords:systemiclupuserythematosus;extracellularvesicles;microRNA;differentialexpression;biologicalfunctioSystemiclupuserythematosus(SLE)isacomplexautoimmunediseasewithdiverseclinicalmanifestations.ThediagnosisofSLEcanbechallengingduetothevariableandnonspecificsymptoms,andthereisaneedforbetterdiagnostictools.Extracellularvesicles(EVs)aresmallmembranousstructuresthatcarrybiomoleculessuchasmicroRNAs(miRNAs)andareinvolvedinintercellularcommunication.Inrecentyears,therehasbeengrowinginterestintheuseofEV-associatedmiRNAsasbiomarkersforvariousdiseases,includingSLE.

Inthisstudy,weanalyzedtheexpressionprofilesofmiRNAsinEVsisolatedfromtheserumofSLEpatientsandhealthycontrols.WefoundthatseveralmiRNAs,includingmiR-146a,miR-155,andmiR-21,weredifferentiallyexpressedinEVsfromSLEpatientscomparedtocontrols.Interestingly,theexpressionlevelsofthesemiRNAswereassociatedwithspecificclinicalfeaturesofSLE,suchasdiseaseactivityandrenalinvolvement.

MiR-146ahasbeenimplicatedinnegativeregulationofinflammation,anditsdownregulationinSLEEVsmaycontributetothechronicinflammatorystateseeninthisdisease.MiR-155hasbeenshowntobeinvolvedinimmunecellactivationanddifferentiation,anditsupregulationinSLEEVsmayreflectthedysregulationoftheimmunesysteminthisdisease.MiR-21isinvolvedinfibrosisandtissuerepair,anditsupregulationinSLEEVsmaysuggestaroleinthetissuedamageandscarringseeninSLE.

Overall,ourstudyprovidesfurtherevidenceforthepotentialuseofEV-associatedmiRNAsasbiomarkersforSLEdiagnosisandprognosis.MoreresearchisneededtounderstandthefunctionalrolesofthesemiRNAsinSLEpathogenesisandtodeveloptargetedtherapiesbasedontheirregulationInadditiontotheirpotentialasdiagnosticandprognosticbiomarkers,EV-associatedmiRNAsarealsobeingexploredastherapeutictargetsinSLE.ModulationofmiRNAexpressionlevelscouldpotentiallyaltertheimmuneresponseandreduceinflammationinSLEpatients.SeveralpreclinicalstudieshavedemonstratedtheefficacyofmiRNA-basedtherapiesinmousemodelsofSLE.Forexample,treatmentwithmiR-150antagomirs(whichinhibitmiR-150function)reducedBcellactivationandautoimmuneresponsesinalupus-pronemousemodel(37).

OtherstudieshaveinvestigatedtheuseofexogenousmiRNAstomodulateimmuneresponsesinSLE.Forexample,administrationofmiR-146a,anegativeregulatorofinflammation,reduceddiseaseseverityinamousemodeloflupusnephritis(38).Similarly,intravenousinjectionofmiR-34ainhibitorsreducedrenalinflammationandfibrosisinalupus-pronemousemodel(39).

Morerecently,researchershavealsobeguninvestigatingthepotentialofEVsasdeliveryvehiclesformiRNA-basedtherapiesinSLE.EVscanprotectmiRNAsfromdegradationanddeliverthemtotargetcells,allowingfortargetedmodulationofgeneexpression.Forexample,onestudydemonstratedthatexosomesderivedfrommesenchymalstemcells(MSCs)overexpressingmiR-21couldreduceinflammationandpromotetissuerepairinalupus-pronemousemodel(40).AnotherstudyshowedthatexosomescontainingmiR-146acouldtargetmacrophagesandreduceinflammationinamousemodelofSLE(41).

Whilethesestudiesarepromising,therearestillseveralchallengesthatmustbeovercomebeforemiRNA-basedtherapiescanbeusedinSLEpatients.OnemajorchallengeistheneedfortargeteddeliveryofmiRNAstospecificcelltypeswithintheimmunesystem.EVshaveshownpromiseinthisregard,butmoreresearchisneededtooptimizetheirtherapeuticefficacyandminimizeoff-targeteffects.

Inconclusion,EV-associatedmiRNAshaveemergedaspromisingbiomarkersandtherapeutictargetsforSLE.ThesesmallregulatoryRNAsplayimportantrolesinimmuneregulationandcontributetothepathogenesisofSLE.FurtherresearchintothefunctionalrolesofthesemiRNAsinSLEpathogenesisandthedevelopmentoftargetedtherapiesbasedontheirregulationcouldpotentiallyleadtonewandmoreeffectivetreatmentsforthiscomplexautoimmunediseasePotentialareasforfutureresearchonEV-associatedmiRNAsinSLEinclude:

1.IdentificationofnovelmiRNAs:WhileseveralmiRNAshavealreadybeenidentifiedaspotentialbiomarkersortherapeutictargets,theremaybeothermiRNAsthatarespecifictocertainsubsetsofSLEpatientsorthathaveyettobediscovered.AdvancedsequencingtechniquescouldbeusedtoidentifyandcharacterizenewmiRNAsassociatedwithSLE.

2.Mechanismsofaction:WhilesomeofthefunctionalrolesofEV-associatedmiRNAsinSLEhavebeenelucidated,manyquestionsremainregardingthedownstreamtargetsandpathwaysregulatedbythesemiRNAs.FurtherexplorationofthemolecularmechanismsthroughwhichthesemiRNAsexerttheireffectscouldprovideinsightintonoveltherapeuticstrategies.

3.Clinicalcorrelation:ManystudieshaveshownassociationsbetweencertainmiRNAsandSLEdiseaseactivityorspecificclinicalmanifestations.However,itremainsunclearwhetherthesemiRNAsarecausallyrelatedtothediseaseorsimplybiomarkersofdiseasestatus.LongitudinalstudiesthatfollowpatientsovertimeandincludelargersamplesizescouldhelptoestablishcausalrelationshipsbetweenspecificmiRNAsandSLEmanifestations.

4.Developmentoftargetedtherapies:TargetingspecificmiRNAsortheirdownstreampathwayscouldbeapromisingtherapeuticapproachforSLE.However,developingsafeandeffectivemiRNA-basedtherapieswillrequirecarefulconsiderationofoff-targeteffectsandpotentialtoxicity.PreclinicalstudiesinanimalmodelsandphaseIclinicaltrialswillbenecessarytoassesssafetya