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miRNAINPATHOBIOLOGY(TPATEL,SECTIONEDITOR)MicroRNAandEpigenetics:DiagnosticandTherapeuticOpportunitiesPalomadelC.
MonroigGeorgeA.
CalinPublishedonline:19January2013SpringerScience+BusinessMediaNewYork2013AbstractMicroRNAs(miRNAs)arealargefamilyofpost-transcriptionalregulatorsofgeneexpressionthatcontrolcellularanddevelopmentalprocessesbytargetingmessengerRNAs.
Thesesmallnon-codingRNAs(ncRNAs)areaber-rantlyexpressedincancerandareknowntocontributetotumorigenesisanddiseaseprogression.
TherapeuticstrategiesbasedonmodulatingmiRNAsactivityareemergingduetotheabilityofthesencRNAstoinuencecellularbehavior.
MiRNAlevelspredictdiseaseprognosisandoverallpatientsurvival,andreconstitutingtheirbasallevelshasbeenproventoinhibittumorgrowthandmetastasis.
Differentdeliverymechanismshavebeentestedinvivo,howevermanychal-lengesneedtobeovercomebeforetheirutilizationintheclinic.
Moreover,ithasbeenfoundthatcirculatingmiRNAsinbodyuidshavethepotentialtoreshapecancerdiagnosisandprognosisbyfunctioningasbiomarkersandindicatorsofprogressionandmetastasis.
ThesemiRNAsasbiouids-basedbiomarkersprovideanalternativestrategyforearlydiagnosisandtreatmentofcancerpatients.
KeywordsMicroRNAEpigeneticsCancerDiagnosisTherapyBiomarkersPathobiologyIntroductionMicroRNAs(miRNAs)aresmallnon-codingRNAs(ncRNAs)of*22ntwhichpost-transcriptionallyregulategeneexpression.
ThemajorityofthesesmallRNAmole-culesaretranscribedbyRNApolymeraseIIasindependentgenes,orasintronsofprotein-codinggenes[1].
MiRNAstargetmessengerRNAs(mRNAs)causingcriticalchangesandinstabilitythatconsequentlyinhibittranslation.
Apercentageofapproximately60%ofgenesinthehumangenomearepredictedtobetargetedbymiRNAs,astheyconservepairinghomology[2].
MechanismsofActionMiRNAsareinitiallytranscribedbyRNApolymeraseIIenzymeaslongprimarytranscripts(pri-miRNAs).
Thistranscriptfoldsonitselfformingadoublestrandedhairpinstructure.
AnRNaseendonucleaseIIIDrosha,alongwithDGCR8(molecularanchorpartofamicroprocessorcom-plex),cleavesthissecondarystructure,resultingin*70ntprecursormiRNAs(pre-miRNAs).
Exportin5andRAN-GTPpromotenucleartranslocationofthepre-miRNAstothecytoplasm.
Dicer,anotherdoublestrandedRNA-spe-cicnucleaselocatedinthecytoplasm,denesacleavagesitethatresultsin*22ntlongdoublestrandeddsRNAcontainingamaturemiRNAguidestrandandapassengerstrand[3].
TheguidestrandpromotestheassociationofaproteincomplextermedRNA-inducedsilencingcomplex(RISC)withthe30untranslatedregionofthetargetedmRNAs.
TheprimarycomponentoftheRISCcomplexisanArgonaute(Ago)protein,whichcausemiRNA-medi-atedmRNArepression.
TheAgoproteiniscrucialforthemiRNAtoformacomplexwithRISC;moreover,itallowsmiRNAmaturationevenintheabsenceofDicerbyacquiringtheabilitytocleavepre-miRs.
ThisevidencewasidentiedbyChelouetal.
,whoobservedthatmiR-451couldenterRISCthroughanalternativebiogenesispath-way.
AfterDrosha-mediatedcleavage,thepre-miRwasP.
d.
C.
MonroigG.
A.
Calin(&)DepartmentofExperimentalTherapeutics,UniversityofTexasM.
D.
AndersonCancerCenter,Houston,TX77030,USAe-mail:gcalin@mdanderson.
orgP.
d.
C.
Monroige-mail:Paloma.
D.
Monroig@uth.
tmc.
edu123CurrPathobiolRep(2013)1:43–52DOI10.
1007/s40139-013-0008-9loadeddirectlyintoAgoomittingthecatalyticactivityofDicerandformingacomplexwithRISC[4].
RolesandGenomicRegulationMiRNAsregulatetheexpressionofspecictargets.
How-ever,sincethenucleotidepairingbycomplementarityofthesesequencesisimperfect,itallowskeymiRNAstoregulatetheexpressionlevelsofhundredsofgenessimultaneously[5].
MiRNAprolesindifferenttissuesaresubjecttoregulatingmechanismsthatincreaseordecreasetheirdownstreameffect(s).
Thismechanismofactionimplicatestheminvariousimportantprocessessuchastissuemorphogenesis,apoptosisandsignaltransductionsimultaneously[6,7].
This,alongwithotherevidence,hasprovedthatmiRNAexpressionsignaturesareassociatedwithhumanpathologiessuchascancer.
MiRNAsaresubjecttogenomicregulation,whichcanbesegregatedinfourgroups:amplication,deletions,mutations/translocations,andepigeneticalterations[8].
AllelicamplicationofmiRNAsresultsindecreasedexpressionofthetargetgene,proportionaltomiRNAlevelsinthecell.
Genomicdeletiondecreasestheexpres-sionofmiRNAs,increasingthegeneticexpressionofthetargetedgenes.
Translocationsandsequencevariations,suchasmutationsorsinglenucleotidepolymorphisms(SNPs),canalterthetranscribedmiRNAs,causingchangesinthebindingsitesandinhibitingorpromotingrepression.
Finally,epigeneticalterationssuchasmethylationofthepromoterregionalsocausechangesinthemiRNAsthatmatureinacell,decreasingtheirbasallevels[8](Fig.
1).
TherapeuticOpportunitiesinCancerMiRNAsnegativelyregulategenesinvolvedineukaryoticsurvivalandproliferation,andcancauseadisturbanceinproteinexpression,translationandstability.
TherstassociationsobservedbetweenmiRNAsexpressionandcancerdevelopmentwasthemiR-15andmiR-16dysreg-ulationinBcellchroniclymphocyticleukemia,asaresultofchromosome13q14deletion[9].
ManymiRNAgenesarelocatedinfragilegenomicsites(regionsmoresuscep-tibletomutations,rearrangementsandlossofheterozy-gosity),whicharefrequentlyfoundintumordevelopment[10].
AlterationofproteinsinvolvedinmiRNAregulationandthetargetingofimportantmessengerRNAs(mRNAs)areinvolvedintumorinitiationandprogression,asthesecodinggenesbehaveasoncogenesortumorsuppressorgenes[11].
TumorsuppressivemiRNAsfunctionbyreducingtheexpressionofproto-oncogenes,delayingcar-cinogenesis,andtumormaintenance.
ThemiR-200familyisanexampleofatumorsuppressivemiRNA,proventoinuencetheepithelialphenotypeofcellsbydownregu-lationofE-cadherin(promotingepithelial-to-mesenchymaltransition)[12].
Ontheotherhand,oncogenicmiRNAsreduceexpressionoftumorsuppressorcodinggenesandareover-expressedorampliedintumorcells,contributingtotumordevelopment.
SomeoncogenicmiRNAsinhibitrepressors,causinganincreaseintranscriptionfactorsorgrowthfactors(e.
g.
,R-Smads,ER-a,p53)andauxiliaryproteins(e.
g.
,hnRNPA1).
InthiswaytheyinuencethemRNAprocessingmachineryandcausealterationsintheirbasallevels[13].
ThedysregulationofmiRNAsincells,andthesubsequentgain/lossoffunctions,dictatetreatmentefcacy,therapeuticresponse,andpatientprognosis.
Twoimportantstrategieshaveguidedtheuseofthesemoleculesasnoveltherapies.
First,miRNAmoleculeshavebeenmodiedtoachieveaprolongedinvivohalf-lifeandefciency(e.
g.
,anti-miRNAandlockednucleicacid(LNA)-modiedoligonucleotides,andantagomirs).
Sec-ond,invivodevelopmentofmiRNAtransgenicmice(suchasthemiR-155,miR-21,miR17–92)andknockouts(suchasmiR-15,miR-16,miR-146andmiR-29)haveofferedvaluableinformationthathasguidedtherapeuticopportu-nitiesforcancerpatients[8].
Thestrategiesarebasedontwoprinciples:targetingoncogenicmiRNAs(todecreasetheirlevels),orrestoringtumorsuppressivemiRNA(torescuetheirbasallevels).
TargetingOncogenicmiRNAsCancercellscontainmanygeneticandepigeneticabnor-malities,butdespitetheircomplexity,theirgrowthandsurvivalcanoftenbeimpairedbyinactivatingasingleoncogene.
Thisphenomenon,called''oncogeneaddiction,''providesarationaleformoleculartargetedtherapy[14].
CorrelationsbetweenregulatorymiRNAsandcancerhaverevealedthatthisconceptappliestomiRNAdysregulationinpatients.
TherapiesagainstoncogenicmiRNAfocusondecreas-ingmiRNAlevelsbyinhibitingthemthroughcomple-mentarybasepairing.
AlthoughinhibitinganindividualmiRNArolewastheinitialstrategy,studieshavehigh-lightedtheimportanceoftargetingthe''collaborativerole''ofmiRNAgroupsasthesecooperateintheacquisitionofapleiotropicbiologyincancercells.
Bothoftheseapproa-cheshavebeenpointedoutinmiR-17–92,anoncogenicpolycistronicallyexpressedclustercomposedofsixmiR-NAs:miR-17,-18a,-19a,-20a,-19b-1and-92a-1onchromosome13.
OverexpressionofthemiR-19familymembers(relativetotheircluster),provedtoincreasethelatencyoflymphomas,whiletheirinactivationpromotedMYC-inducedlymphomagenesis[15].
BothoftheseresultssuggestanimportantindividualroleofthemiR-19family.
Furthermore,multiplemembersoftheclusterarecapable44CurrPathobiolRep(2013)1:43–52123ofindividuallypromotingNOTCH1-inducedTcellacutelymphoblasticleukemiainamousemodel[16].
Thesend-ingsontheotherhandemphasizethecollaborativeroleofthreemiRNAsofthecluster(miR-19b-1,-20aand-92a-1)inreducingdiseaselatencybydecreasingtheexpressionofthetumorsuppressorsPTENandBIM(typicallydown-regulatedinT-ALL)[16].
BecauseofthediversityofmechanismsbywhichmiRNAlevelscontributetotumorinitiationandprogression,severaltherapeuticmodelshavebeendevelopedtotargettheseprocesses.
TherapiestodecreasetheeffectofaspecicmiRNAhavebeenproposedbyusingantisenseoligonucleotides(ASOs)thatinhibittargetsbyadheringtothemiRNA,formingmiRNA-anti-miRNAbindingcomplexes.
Threetypeshavebeendescribed:antagomirs,LNAsandASOswithchemicalalterationstooptimizeefcacy(AMOs)[17,18,19](Table1).
WiththeuseofASOs,Fontanaetal.
[20]demonstratedthattumorgrowthwasinhibitedwheninjectingtheantagomir-17-pintherapyresistantneuroblas-tomacelllines.
Inreferencetothechemicallymodiedforms,Maetal.
[21]usedAMOstointravenouslyinhibitmiR-10binamammarymousetumormodel,andobservedthatmetastasiswasinhibited.
Parketal.
testedanotherchemicallymodiedantagomir,chol-anti-miR-221.
Intheirorthotopicmousemodelofhepatocellularcarcinoma,theyprovedthatchol-anti-miR-221signicantlyreducedmiR-221levelsintheliver,andthatthis,correlatedwithareductionintumorcellproliferation,anincreaseinapoptosismarkersandcellcyclearrest[22].
LNA's,anothertypeofASOhavealsoproventobeefcient,asaliverregenerationmousemodeltestedbySapraetal.
[23],demonstratedthatusingalockednucleicacidASOagainstsurvivin(anapoptosisinhibitor),reduceditsmRNAlevelsin80%.
Researchershaverecentlyengineeredasinglesubunittermed''multiple-targetanti-miRNAantisenseoligode-oxyribonucleotide''(MTg-AMO),throughwhichsimulta-neoussilencinghasbeenachieved[24].
MTg-AMO'shavebeenproventoallowtherestorationofdysregulatedmiRNAlevelsbytargetingseveralkeyaspectsofthebiologyofcancercellsintumortissueatonce.
Luetal.
,demonstratedthattheMTg-AMOtargetingmiR-21,miR-155andmiR-17-5pproducedagreaterinhibitoryeffectoncancercellgrowth,comparedwiththeregularsingle-targetAMOs[24].
AnothertherapeuticapproachtodecreasemiRNAlevelsinvolvesexpressingcompetitiveinhibitorsoftheirfunction.
Fig.
1MicroRNAlevelsarealteredincancerthroughdifferentmechanisms:(a)amplicationofoncogenicmiRNAsresultsindecreasedexpressionoftargetgenes,(b)mutationscandecreasetheexpressionoftumorsuppressivemiRNAsorcouldpotentiatetheexpressionofoncogenicones,(c)epigeneticchangessuchaspromotermethylationortrans-actingfactorschangetheexpressionproleofmiRNAsinacellbydecreasingorincreasingtheirbasallevels,(d)deletionseliminatetheexpressionoftumorsuppressivemiRNAsincreasingtheexpressionoftargetgenes.
TherapeuticstrategiestargetthesealterationsandaimtorestoremiRNAbasallevelsCurrPathobiolRep(2013)1:43–5245123Atypeoftheseare''microRNAsponges''whicharevectorscontainingmultiplearticialmiRNAbindingsitesthatareplacedunderthecontrolofstrongpromoterstoproducelargequantitiesoftranscript[25].
ThesetranscriptsexpressmultipletandemsitestoanmiRNAofinterest[26].
MiRNAspongeswereusedinmetastaticbreastcancermousemodel,whereinvivoexperimentsdemonstratedthatthedownregulationofanover-expressedbreastcancermiRNA(miR-19)couldbeachieved,alongwithmetastasicinhibi-tion[27].
ApproacheshavealreadybeendevelopedtorapidlygeneratesingleorcombinedmiRNAspongesthatcanbeusedforlong-termmiRNAloss-of-functionstudies[28].
RestoringExpressionofTumorSuppressivemiRNAsAlthoughspecicmiRNAsareoverexpressedincancertissue,severalmiRNAsaredownregulatedintumors[29].
GlobalrepressionofmiRNAexpressionhasbeenproventoincreasetumorigenesisinbothinvitroandinvivomodels[30].
Thedownregulationofelementsofthebiogenesispathway(processingmachinery),isoneofthemechanismsbywhichmaturemiRNAlevelsarereducedinsometypesofcancer.
AnexampleofthiswasseenbyMerritetal.
whentheystudiedDicerandDroshalevelsinpatientswithovariancancer.
TheyfoundthatpatientswithalowerexpressionweresignicantlyassociatedwithlowmiRNAlevels,aswellasadvancedtumorstageandsuboptimalsurgicalcytoreduction[31].
FortumorswithreducedexpressionofmiRNAs,resto-rationoftheirbasallevelsisthekeystrategy,whichcanbeachievedthroughmiRNAmimeticsorbyregenerating''miRNAome''(fullspectrumofexpressedmiRNAsinacellataspecictime)functionality(Table2).
ThemostwidelyusedapproachisusingmiRNAmimeticswhicharesyntheticsmallRNAsthatcontaintheexactsequenceoftheendogenousones.
Toachievethedeliveryofastablemolecule,miRNA'saredeliveredasperfectlycomple-mentaryduplexes,similarinarchitecturetosiRNAs[32].
Themajorityofthetherapiesthathavetriedtorestoretumor-suppressivemiRNAwithmimeticshaveachievedtheirgoalbyadministeringthemlocally.
However,nowa-daysthechallengeofdevelopingasystemictherapyinatissue/cell-typespecicmannerhasbeenproventobeachievablethroughdifferentdeliverymechanisms.
Ibrahimetal.
[33]usedbothsystemicandlocalapplicationoflowmolecularweightpolyethylenimine/miR-145andmiR-33acomplexeswhichweredeliveredintomousexenograftTable1InhibitorymiRNAdrugsAgentWhataretheseagentsMechanismofactionExampleAntagomirsSingle-stranded23ntRNAmoleculescomplementarytothetargetedmicroRNAthathavebeenmodiedtoincreasethestabilityoftheRNAandprotectitfromdegradation.
Themodicationsincludedapartialphosphorothioatebackboneinadditionto20-O-methoxyethylThemechanismisnotcompletelyknown,howeveritisbelievedtoactbyformingaduplex:(miRNA/antagomir)thatinducesthedegradationofthetargetedmiRNAandrecyclingoftheantagomirAntagomir-17-5p:ShowntoabolishthegrowthofMYCN-ampliedandtherapy-resistantneuroblastomaleadingtocellcyclingblockadeandapoptosis[20]AMOsanti-miRsSingle-strandedASOs,chemicallymodiedtoenhancebindingafnitytoacomplementarymicroRNA.
UsuallyshorterthantheirtargetmiRNA*17–20ntinlengthandinhibitsinaspecicmannerthegeneticexpressionofitstargetgeneProduceanASO-miRNAdoublestrandedcomplexthroughcomplementarybasebinding,leadingtonon-specicendonucleasecleavageofthetargetedmiRNAAnti-miR-10b:Chemicallymodiedanti-miRNAoligonucleotideshowntosuppressbreastcancermetastasisinmousemammarytumormodel[21]LNAanti-miRsAnucleicacidanaloguecontainingoneormoreLNAnucleotidemonomerswithabicyclicfuranoseunitlockedinanRNAmimickingsugarconformationSameastheAMOEZN-3042:ALNA-ASOproventodownregulatesurviving(apoptosisinhibitor),andtherebyinhibittumorgrowthinvivo[23]MTg-AMOsModiedAMO,inwhichmultipleantisenseunitsareengineeredintoasingleunitSameastheAMO,exceptthatthemultiplicityofunitsenablesittosimultaneouslysilencemultipletargetmiRNAsMTg-AMOagainstmiR-21,miR-155andmiR-17-5p:Producedagreaterinhibitoryeffectoncancercellgrowth,comparedwiththeregularsingle-targetAMOs[24]MicroRNAspongesTranscriptsthatcontainmultipletandem-bindingsitestoamiRNAofinterestandaretranscribedfromexpressionvectorsBycompetingwiththenativetargetsofmiRNAs,thesehighlyexpressedtranscriptsresultinincreasedexpressionofthemiRNA'snativetargetsmiRNAspongetoincreasemiR-9targeting:Metastasisformationwasinhibitedinhighlymalignantbreastcancercells[25]46CurrPathobiolRep(2013)1:43–52123tumorsofcoloncarcinoma,obtainingantitumoreffectssuchasreducedproliferationandincreasedapoptosis.
Inanothermodel,thesystemicadministrationofmiR-26athroughanadenoviralvectorwasgiventomicewithhepatocellularcarcinoma,resultingintheinhibitionofcancercellproliferation,inductionoftumorcellapoptosisandprotectionfromdiseaseprogression[34].
Averyrecentorthotopicmodelforhepatocellularcarcinomaachievedahighertherapeuticefcacythanpreviousones;consistentlydecreasedlevelsofmiR-34awheretargetedwithliposomaldeliveryofthemiRNAinarelevantNOV340/miR-34aformulation.
Theeffectivenessofdecreasingtumorigenesisinthismodelhasledtheresearcherstoanticipatetheini-tiationofclinicaltrialsin2013,withmiR-34abeingoneoftherstmiRNAmimicstoreachtheclinic[35].
Alongwithmimetics,othertherapieshaveaimedatrestoringmiRNAlevelsina''globalmanner''.
TwoofthesetherapiesareepigeneticmodiersandenhancersofthemiRNAprocessingmachinery.
Epigeneticmodiersaddresschangessuchashyper-methylationandhistonemodicationpatterns,whichhavebeenlinkedtomiRNAdysregulation.
SaitoandcolleaguesaimedattargetingthehypermethylationthatsilencedthetumorsuppressormiR-127inhumancancercells.
Forthis,theyusedchromatin-modifyingdrugssuchas5-aza-20-deoxycytidineand4-phenylbutyricacidinhumancancercells.
AfterthetreatmenttheyobservedarestorationinmiR-127levelsandsubsequentinactivationofBCL6proto-oncogene[36].
Otherresearchgroupshavefurther-morecombinedstrategiesusingDNAdemethylatingagentsandhistonedeacetylaseinhibitors.
HuangandcolleaguestestedboththerapiestoregeneratebasallevelsoftumorsuppressormiR-129-2.
TheirtreatmentresultedinthereactivationofmiR-129-2incancercells,withthedownstreameffectofdecreasingSOX4(transcriptionfac-tor)levelsandreducingproliferationinendometrialcancercells[37].
ThedisruptionofthemiRNAprocessingmachineryhasalsobeenknowntocontributetothedevelopmentofhumantumors,aspreviouslymentioned[31].
Scientistshaverecentlycomeupwithasmallmolecule/drugthatenhancesRNAiandpromotesmiRNAprocessing:Enox-acin[38].
ThisisauoroquinolonethatincreasestheproductionoftumorsuppressivemiRNAbybindingtothetransactivation-responsiveRNA-bindingprotein2(TRBP).
Inhumanculturesandametastaticmousemodel,theuseofthisdrughadacancer-specicgrowth-inhibitoryeffect[38].
TheseresultssuggestthatnovelstrategiesaimingtorestorethemiRNAomemightbepromising.
DeliveryMechanismsInvivodeliveryisthemainfocusofmiRNAbasedther-apies,andeitherlocalorsystemictechniqueshavebeendeveloped.
Localstrategies,suchasdirectsiteinjection,haveseveraladvantagesastheyevadenucleasedegrada-tion,decreasemiRNAmodulatoruptakebynon-targetedtissuesandincreasebioavailabilityintumorsites.
Never-thelesstheyaremerelyapplicabletoaverylimitedlistoftargettissuessuchasocular,brain,sarcomas,mesothelio-mas[39];andeveninthesetissuesnotalltargetedtumorcellscanbereachedbythemiRNAmodulators[40].
Thesecondapproach,asystemicdelivery,consistsofabloodstreaminjectionthat—intheory—shouldhaveamuchmoreefcientdisseminationtothetargettissues.
However,thesemechanismsneedtoovercomeinvivobarrierssuchasnucleasedegradationandnon-specicTable2DrugsthatrestoremiRNAAgentWhataretheseagentsMechanismofactionExamplesmiRNAmimeticsSyntheticsmallRNAsthatcontaintheexactsequenceofendogenousmiRNA.
TheycanbemodiedtohaveenhancedefciencybyincreasingtheafnityforaspecictargetandreducingotherunwantedmiRNAeffectsDeliveredasperfectcomplementaryduplexesandtheyactbyimprovingtheRISCloadingofmiRNAmiR-26a:Inahepatocellularcarcinomamousemodelthismimeticinhibitedcellgrowthandapoptosis[34]EpigeneticmodiersMoleculesthatreverseDNAmethylationandposttranslationalhistonemodicationsEitherinhibitmethylation,orinteractwithenzymesthatremoveacetylgroups5-aza-20-deoxycytidineand4-phenylbutyricacid:RestoredtumorsuppressivemiR-127levelsandsubsequentlyinactivatedBCL6proto-oncogene[36]EnhancersofmiRNAprocessingmachinerySmallmolecule/drugthatenhancesRNAiandpromotesmiRNAprocessingIncreasetheproductionoftumor-suppressivemiRNAyinteractingwiththebindingproductsoftheproteinsthatregulatemiRNAmaturationEnoxacin:AuoroquinolonethathasbeenproventoincreasetheproductionoftumorsuppressivemiRNAbybindingtothetransactivation-responsiveRNA-bindingprotein2(TRBP)[38]CurrPathobiolRep(2013)1:43–5247123targetingofallthetissuesthatarereachedbybloodves-sels,specicallytheliver,jejunum,lungandkidney[40].
Chemicalmodications,encapsulationsandconjugationshaveaimedtoprotectthesetherapeuticmiRNAs,becauseonceinthebloodstreamtheyarerequiredtomaintainintact.
Conservingtheirstructureallowsthemtotraversecellularmembranesandmoreimportantlyreachthecytosoloncereleasedfromtheirendosomevesicle[41].
CarriersSynthesizingastable,biodegradableandbiocompatiblemiRNAmodulatorisveryimportant,butensuringanadequatecellularpenetrationofthedeliverymoleculeisalsocrucial.
Carrierscanbedividedintwogroups:viralandnon-viral,andbothtypesaimtotargettumorsystemswhilesimultaneouslyevadingimmunetoxicity.
Viralstrategieshavebeenusedwithvectorssuchaslentiviruses,adenovirusesandadeno-associatedviruses(AAVs),andgeneticmanipulationcantargettheseentitiestothecellsofchoice[42,43].
Althoughpreclinicalmousemodelsusingvirusasvectormediateddeliveryhaveshownpromisingresults,theypresentsomeawsthatstillneedtobeaddressedbeforeadvancingintotheclinicalpractice.
Someofthestrategiestoovercometheseawscouldfocusonobtainingefcientnuclearlocalization,evadingtheactivationofoncogenesorinactivationoftumorsuppressorgenes,andeliminatingundesiredimmunogenicresponsesinpatients[41].
Non-viralstrategiessuchasliposomaldeliveryhavebeenwidelyused,andtheyhavebeendemonstratedtoinducetumorsuppressioninseveralmicemodels[44–46].
Theirsuccesshasbeenattributedtotheprotectionofoli-gonucleotidesfromnucleasemediateddegradation,alongwiththeirabilitytoincreasethecirculatinghalf-lifewhensystemicallydelivered[39].
However,invivosystemshavehadadverseeffectsrelatedtothepositivechargeofthelipidcomponent[39,47].
Onestrategytoovercomethesetoxiceffectsisdevel-opmentofneutralnano-liposomesbasedon1,2-dioleoyl-sn-glycero-3-phosphatidylcholine(DOPC)[48].
Thesenano-liposomescandelivermiRNAinvivointotumorcells10-and30-foldmoreeffectivelythancationiclipo-somes[49],andhavebeenproveninmousemodels.
Forexample,inamousemodelforlungcancer,Trangetal.
[50],systemicallydeliveredthetumorsuppressormiR-34ainaneutrallipidemulsionandobtaineda60%reductionintumorareacomparedtocontrols.
Inanothermodel,micewithovariancancertumorsweretreatedwithsiRNAincorporatedintotheneutral-DOPCliposomestotargettheoncoproteinEphA2.
IntheseexperimentsLandenetal.
[49],observedreducedtumorgrowthwhencomparedwithanonsilencingsiRNA.
Otheralternativesincludecoatingcationicbilayerswithhydrophilicpolyethyleneglycol(PEG),whichalsodecreasesimmunogenicresponses[51].
Finally,theaddi-tionofbiodegradablesyntheticornaturalpolymerscouldalsorepresentaneffectivealternativetofacilitatesustaineddeliveryinvivo(e.
g.
,polyethyleneimines);nevertheless,toxicityremainsachallenge.
BodyFluidExpressedmiRNAs:BiomarkersandTreatment-ResponsePredictorsDifferenttypesofcancershavespecicmiRNAprolesthatallowtumorstobeclassiedbasedontheirmiRNAexpressionlevels.
ThelackofcomplextranscriptionalandtranslationalmodicationscomparedtomRNAsandpro-teinssuggestthattheuseofmiRNAsasbiomarkersforcancerhasgreatpotential.
Sincetheremustbesomemethodofcommunicationbetweencancercellsandtheirmicroenvironment,researchhassuggestedthatmiRNAsshouldbeconsideredmoleculesofsecretionalongwithcytokines,growthfactorsandotherproteins[52].
SerumandplasmacanbothbeusedtomeasurespecicmiRNAlevels,astherecomparisoninseveralstudieshasledtoequaldistributionofmiRNAs.
Mitchelletal.
[53],foundastrongcorrelationwhencomparingbothoftheseinthebloodofmiceinprostatecancerxenograftmodel;more-over,aprolestudydoneinserumandplasmaoflungcancerpatientsledtothesameconclusions[54].
StudieshavealreadydemonstratedcorrelationbetweencirculatingmiRNA-expressionlevelsandresponsetoanti-cancertreatment[55],givingmiRNAacrucialroleinthera-peuticmanagementofcancerpatients.
ResearchgroupshavefocusedonsegregatingcirculatingmiRNAsinspecicpat-ternsbasedonthedifferentcancertypes(foramoredetailedreviewseeAllegraetal.
[56]).
InthisreviewIwillrefertofourofthecancertypeswiththehighestincidence(lung,breast,colonandhematologicmalignancies),andcurrentresearchndingsoncharacteristicuidexpressedmiRNAs.
LungCancerMorepeoplediefromlungcancerthanfromanyothertype,andintheUSover200,000caseswerediagnosedin2012,andapproximately160,000diedfromthedisease[57].
ThishasincreasedresearchincludingstudiesinthemiRNAeld.
Inlungcancer,circulatingmiRNAshavebeenlinkedtopre-diseasepatterns,diseasestaging,tumoractivityandmetastasisdevelopment.
Researchhasrecentlygeneratedahigh-throughputstudy,inwhichforthersttimeitwasdemonstratedthatpre-diseasesignaturesofmiRNAexpressioninplasmasamplescanpredictdevel-opmentoflungcancerbeforeacquiringadiagnosisfrom48CurrPathobiolRep(2013)1:43–52123conventionaltechniques[58].
MiR-21wasrecentlydescribedasamiRNAthatcoulddifferentiateearlystagelungcancerpatientsfromhealthyindividuals;furthermore,itsplasmalevelsnotonlyserveasacirculatingtumorbio-marker,butcouldalsodeterminesensitivitytoplatinum-basedchemotherapy[59,60].
HighcirculatingmiR-155,miR-197andmiR-182werealsocorrelatedwithdiseasestageI,asplasmasamplesfrompatientsdemonstratedsignicantlyhigherlevelsthantheirrespectivecontrols[61].
Finally,miR-486-5phasbeenalsostudiedinplasmaoflungcancerpatients,andatumor-suppressiverolehasbeenassociatedwithitsdownregulation[59,62].
ThesendingssuggestthatspecicmiRNAsserveasmarkersofdiseasestageandaprognosisindicator[61].
BreastCancerBreastcanceristhemostcommoncanceramongAmericanwomen(asidefromskin),andmiRNAshavealsoacquiredsignicanceinthisdisease.
IntheUnitedStatesin2008,210,203womenwerediagnosedwiththedisease,and40,589womendiedfromit[63].
AlteredmiRNAshavebeenstudiedbyseveralgroups:Rothandcolleaguesreportedndingsofalteredlevelsintheserumofbreastcancerpatients[64].
Moreover,Mitchelletal.
[53]andHeneghanetal.
[65]demonstratedtumorigenicrolesofmiR-195andlet-7ainbreastcancer,astheyprovedthatlevelswereincreasedwiththepresenceofthetumor,anddecreasedinserum2weeksaftersurgicalresection.
ColorectalCancer(CRC)Ofcancersaffectingbothmenandwomen,colorectalcanceristhesecondleadingcancerkillerintheUnitedStates[66].
CRChasalsobeenstudiedtondalternativebiomarkersofdiseaseprogression.
Forstagingtechniques,Chengetal.
,foundthatcirculatingmiR-141wassigni-cantlyassociatedwithstageIVcoloncancerinacohortofplasmasamples.
TheyalsoobservedthatcombiningthedetectionofmiR-141andcarcinoembryonicantigen(CRCmarker)furtherimprovedtheaccuracyofdetectionandpatientsthathavebothofthemwereassociatedwithapoorprognosis[67].
OtherpotentialbiomarkersstudiedhavebeenthecirculatinglevelsofmiRs-17-3pand-92.
Thesewereshowntodecreaseaftersurgery,andingthatservedtodifferentiateCRCpatientsinrecoveryfromothergastriccancerpatientsandfromnormalindividuals[68].
HematologicalMalignanciesIntheUSin2009,therewereapproximately271,880menandwomenalivewhohadahistoryofleukemia,andover10%ofthesearediagnosedbeforetheageof20[69].
Inthereachofdiscoveringnewtherapeuticmodalitiesforthesepatients,theroleofmiRNAsisstillbeingelucidated[69].
Inhematologicalmalignancies,miRNAshaveanimportantrole,astheyparticipateinleukemogenesisbycoordinatingeffectswithkeytranscriptionfactorsrequiredforhematopoiesis.
Moussayetal.
studied14miRNAsassociatedwithCLLandfoundthatthreeofthem—miR-195,miR-29-aandmiR-222—stronglyseparatedcontrolsversusCLLpatients.
Moreover,increasedlevelsofmiR-150increasedwithdiseaseseverity,suggestingthepoten-tialofthisbiomarkerindiseasestaging[70].
Inothermalignancies,suchasBcelllymphoma,miRNAssuchasmiR-21,miR-155andmiR-210werefoundtocorrelatewithfavorableclinicaloutcome,astheywereassociatedwithrelapse-freesurvival[71].
AlthoughnotmanystudieshavebeendoneofcirculatingmiRNAinacuteleukemias,recentevidencehavepointedoutthatdecreasedplasmalevelsofmiRNA-92isasignicantbiomarkerforpatientspresentingacutemyeloidleukemia[72].
ConclusionThegeneralideaofreconstitutingthemiRNAomeas''awhole''hasresultedinimprovingstrategiestoachieveareductionintumorgrowthandmetastasisinhibition.
ThedevelopmentofmiRNA-basedtherapies(suchasmolec-ulardrugs,mimeticsandAMOs)hascontinuedtopro-gress,astheinvivomodelsanddeliverymechanismsbeingdevelopedaimtobelesstoxicandmoreefcientinreachingtheirtargets.
Thesynthesisofnanoparticlescomposedofpolymers,neutrallipids,chimerasorcon-jugateswillcontinueimprovinglocalaswellassystemicdelivery.
Nevertheless,severalimmunogenic/nonspeciceffectspointtothenecessityofvalidatingtheefciencyoftherapeuticresponsesinamorepersonalized,patient-specicmanner.
CirculatingmiRNAsnotonlycontributetodiseasestaging,buttheycanimproveprognosisbyemphasizingdiseaseprogressionorregressionandbypredictingmeta-staticpatternsindividuallyinpatients.
CombiningtheuseofknowntherapeuticmechanismswithuidmiRNApro-lescouldresultinanimprovedstrategyagainstcancer.
However,itshouldbetakenintoconsiderationthatmiRNAlevelsintumormicroenvironmentsmightbeacontributionfromcellsthatarenotcancerrelated.
Moreover,othertypesofpathologiescouldalsobegeneratingsimilarmiRNAproles,whichcouldoverlaptheanalysisdoneincancerpatients.
Finally,thefactthatRNA-extractingandquanticationmethods,aswellasnormalizers,varybetweenpublishedstudiesmakeconclusionslesspreciseanduniform.
CurrPathobiolRep(2013)1:43–5249123Biologicaluid-basedmiRNAsproles,alongwithmiRNA-basedtherapies,representanattractiveareaforcurrentcancerresearchbecausetheycouldpotentiallyrevertcancercellsfromtheirtransformedphenotypebacktotheirnormalone.
Inthisway,pharmaceuticaltherapiesareheadedtowardsindividualization,andtheefciencyoftreatmentscouldpotentiallyincrease.
AcknowledgmentsDr.
CalinisTheAlanM.
GewirtzLeukemia&LymphomaSocietyScholar.
HeissupportedalsoasaFellowatTheUniversityofTexasMDAndersonResearchTrust,asaUni-versityofTexasSystemRegentsResearchScholar,andbytheCLLGlobalResearchFoundation.
WorkinDr.
Calin'slaboratoryissupportedinpartbytheNIH/NCI(CA135444),aDepartmentofDefenseBreastCancerIdeaAward,DevelopmentalResearchAwardsinBreastCancer,OvarianCancer,BrainCancer,ProstateCancer,MultipleMyeloma,Leukemia(P50CA100632)andHeadandNeck(P50CA097007)SPOREs,aSINFMDACC_DKFZgrantinCLL,aSINFgrantincolorectalcancer,theLauraandJohnArnoldFoundation,theRGKFoundation,andtheEstateofC.
G.
Johnson,Jr.
DisclosureNopotentialconictsofinterestrelevanttothisarticlewerereported.
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