miR-758 regulates cholesterol efflux through post-

miR-758regulatescholesteroleffluxthroughpost-transcriptionalrepressionofABCA1CristinaM.Ramireza,1,AlbertoDávalosa,1,LeighGoedekea,AlessandroG.Salernoa,NikhilWarriera,DanielCirera-Salinasa,YajairaSuáreza,andCarlosFernández-Hernandoa,2aDepartmentsofMedicineandCellBiology,LeonH.CharneyDivisionofCardiologyandtheMarcandRutiBellVascularBiologyandDiseaseProgram,NewYorkUniversitySchoolofMedicine,NewYork,NY10016,USAAbstractObjective—TheATP-bindingcassettetransporterA1(ABCA1)isamajorregulatorofmacrophagecholesteroleffluxandprotectscellsfromexcessintracellularcholesterolaccumulation,howeverthemechanisminvolvedinposttranscriptionalregulationofABCA1ispoorlyunderstood.WepreviouslyshowedmiR-33wasoneregulator.HereweinvestigatedthepotentialcontributionofothermicroRNAs(miRNAs)topost-transcriptionallyregulateABCA1andmacrophagecholesterolefflux.MethodsandResults—WeperformedabioinformaticanalaysisforidentifyingmiRNAtargetpredictionsitesinABCA1geneandanunbiasedgenome-widescreentoidentifymiRNAsmodulatedbycholesterolexcessinmouseperitonealmacrophages.Quantitativereal-timeRT-PCRconfirmedthatmiR-758isrepressedincholesterol-loadedmacrophages.Underphysiologicalconditions,highdietaryfatexcessinmicerepressedmir-758bothinperitonealmacrophagesand,toalesserextentintheliver.Inmouseandhumancellsinvitro,miR-758repressedtheexpressionofABCA1andconverselytheinhibitionofthismiRNAbyusinganti-miR-758increasedABCA1expression.Inmousecells,mir-758reducedcellularcholesteroleffluxtoapoA1andanti-miR-758increasedit.miR-758directlytargetsthe3′UTRofAbca1asassessedby3′UTRluciferasereporterassays.Interestingly,miR-758ishighlyexpressedinthebrainwherealsotargetseveralgenesinvolvedinneurologicalfunctionsincludingSLC38A1,NTM,EPHA7andMYT1L.Conclusion—WeidentifiedmiR-758asanovelmiRNAthatpost-transcriptionallycontrolsABCA1levelsindifferentcellsandregulatesmacrophagecellularcholesteroleffluxtoapoA1,openingnewavenuestoincreaseapoA1andraiseHDLlevels.KeywordsLipidhomeostasis;microRNAs;atherosclerosisTextCellularcholesterollevelsaretightlyregulatedandrepresentthebalancebetweencholesteroluptake,endogenoussynthesisandefflux.Manydiseasesresultfromperturbationsinlipidhomeostasis,includingatherosclerosis,metabolicsyndrome,typeII2Towhomcorrespondenceshouldbeaddressed:CarlosFernández-HernandoPh.D,522FirstAvenue,Smilow703,NewYork,NY10016,Tel:[phone]Fax:[e-mail](CF-H).1Theseauthorscontributedequallytothiswork.DISCLOSURESNoneNIHPublicAccessAuthorManuscriptArteriosclerThrombVascBiol.Authormanuscript;availableinPMC2012November1.Publishedinfinaleditedformas:ArteriosclerThrombVascBiol.2011November;31(11):2707–2714.doi:10.1161/ATVBAHA.111.232066.NIH-PAAuthorManuscriptNIH-PAAuthorManuscriptNIH-PAAuthorManuscriptdiabetesandAlzheimer'sdisease.Inmammalstwotranscriptionfactorsarerecognizedtothecontrolcellularcholesterolhomeostasis:thesterolresponseelement-bindingproteins(SREBPs)andtheliver×receptors(LXRs).TheSREBPtranscriptionfactorscontrolbothendogenouscholesterolsynthesisanduptakebyregulatingseveralsteroldependentgenesincludingHMG-CoAreductase1,2andthelow-densitylipoprotein(LDL)receptor(LDLR).TheLXRsaremembersofthenuclearreceptorsubfamilyandareactivatedinresponsetocellularcholesterol3.Undercellularcholesterolexcess,LXRtargetsgenessuchastheATP-bindingcassettetransporterA1(ABCA1)andG1(ABCG1),whichpromotecellularcholesteroleffluxandmaintaincellularsterolhomeostasis4,5.Theremovalofexcesscholesterolfromperipheraltissues,suchasmacrophagefoamcells,hasbeenrecognizedasakeymechanismtopreventatherogenesis.6ABCA1promotesmacrophagecholesterolefflux,anddeficiencyormutationsinthistransporterleadstodefectsincholesterolefflux,cholesterolesteraccumulationinmacrophages,andincreasestheriskofdevelopingcardiovasculardiseases7-9.ABCA1alsoplaysanimportantroleinregulatingcholesterolmetabolisminthebrain.Cholesterolisrequiredformyelination,dendritedifferentiationandsynapticactivity.Disturbancesincentralnervoussystem(CNS)cholesterolhomeostasisareimplicatedinneurodegenerativedisorders,includingAlzheimer'sandHuntingtondisease10.SeveralstudieshavedemonstratedthatABCA1facilitatesthecholesteroleffluxofCNScholesteroltoapoE,astheabsenceofABCA1compromisesapoEsecretionfrombothastrocytesandmicroglia11,12.Moreover,theapoEthatispresentinthecerebrospinalfluid(CSF)ofABCA1-deficientmiceispoorlylipidated11.Interestingly,inamyloidmousemodelsofAlzheimer'sdisease(AD),ABCA1deficiencyexacerbatesamyloidogenesis,whereasABCA1overexpressionamelioratesamyloidplaqueload,suggestingaroleforABCA1inAβmetabolism13,14.Inadditiontoclassicaltranscriptionregulators,aclassofnon-codingRNAstermedmicroRNAs(miRNAs),hasemergedascriticalregulatorsofgeneexpressionactingpredominantlyatthepost-transcriptionallevel15.Thislargefamilyofshort(22nt)double-strandedregulatorynon-codingRNAsisencodedinthegenomeandeachmemberisprocessedfromprimarytranscriptsbythesequentialactionsofDroshaandDicerenzymes.UponincorporationintothecytoplasmicRNA-inducedsilencingcomplex(RISC),miRNAsbindtopartiallycomplementarytargetsitesinmessengerRNA