Contents lists available atScienceDirect
Seminars
in
Cell
&
Developmental
Biology
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / s e m c d b
Review
Non-coding
RNAs
as
a
new
dawn
in
tumor
diagnosis
Anna
Grimaldi
a,1,
Mayra
Rachele
Zarone
a,1,
Carlo
Irace
b,1,
Silvia
Zappavigna
a,
Angela
Lombardi
a,
Hiromichi
Kawasaki
a,c,
Michele
Caraglia
a,∗,
Gabriella
Misso
a,∗aDepartmentofBiochemistry,BiophysicsandGeneralPathology,UniversityofCampania“LuigiVanvitelli”,ViaL.DeCrecchio7,80138Naples,Italy bDepartmentofPharmacy,UniversityofNaples“FedericoII”,ViaD.Montesano49,80131Naples,Italy
cWakunagaPharmaceuticalCo.LTD,4-5-36Miyahara,Yodogawa-ku,Osaka532-0003Japan
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received23May2017
Receivedinrevisedform19July2017 Accepted21July2017 Availableonlinexxx Keywords: Non-codingRNA Biomarker Cancer Diagnosis Exosome Delivery
a
b
s
t
r
a
c
t
Thecurrentknowledgeaboutnon-codingRNAs(ncRNAs)asimportantregulatorsofgeneexpressionin bothphysiologicalandpathologicalconditions,hasbeenthemainengineforthedesignofinnovative platformstofinalizethepharmacologicalapplicationofncRNAsaseithertherapeutictoolsoras molecu-larbiomarkersincancer.Biochemicalalterationsofcancercellsare,infact,largelysupportedbyncRNA disregulationinthetumorsite,which,inturn,reflectsthecancer-associatedspecificmodificationof cir-culatingncRNAexpressionpattern.Theaimofthisreviewistodescribethestateoftheartofpre-clinical andclinicalstudiesthatanalyzetheinvolvementofmiRNAsandlncRNAsincancer-relatedprocesses, suchasproliferation,invasionandmetastases,givingemphasistotheirfunctionalrole.Acentralnode ofourworkhasbeenalsotheexaminationofadvantagesandcriticismscorrelatedwiththeclinicaluse ofncRNAs,takingintoaccountthepressingneedtorefinetheprofilingmethodsaimedatidentifynovel diagnosticandprognosticmarkersandtherequesttooptimizethedeliveryofsuchnucleicacidsfora therapeuticuseinanimminentfuture.
©2017ElsevierLtd.Allrightsreserved.
Contents
1. Introduction...00
2. Biogenesisandfunctionalroleofnon-codingRNAs ... 00
3. Mechanismandclinicalrelevanceofexosome-mediatedmiRNAsecretion ... 00
4. ncRNAdysregulationincancer:preclinicalstudiesandtherapeuticimplications...00
5. PotentialofncRNAsasnovelbiomarkersforsolidandhematologicmalignancies...00
5.1. miR155...00 5.2. miR-29...00 5.3. miR-17-92family...00 5.4. NEAT1...00 5.5. MALAT-1 ... 00 5.6. HOTAIR...00 5.7. DLEU1/DLEU2...00
6. miRNAdetectionandquantificationinbodyfluids...00
7. Challenges,opportunitiesandpitfallsofmiRNAprofiling...00
8. Conclusions...00
Conflictofinterest...00
References...00
∗ Correspondingauthors.
E-mailaddresses:michele.caraglia@unicampania.it,michele.caraglia@fastwebnet.it(M.Caraglia),gabrymisso@yahoo.it,gabriella.misso@unicampania.it(G.Misso). 1 Theseauthorscontributedequallytothiswork.
http://dx.doi.org/10.1016/j.semcdb.2017.07.035 1084-9521/©2017ElsevierLtd.Allrightsreserved.
1. Introduction
In the last years it has become increasingly clear that the mammaliantranscriptomeisextremelycomplexduetothe inclu-sionofalargenumberofsmallnon-codingRNAs(sncRNAs)and longnoncodingRNAs(lncRNAs).Non-codingRNAs(ncRNAs)are a heterogeneousclass oftranscribed RNA moleculesfrom non-(protein)-codingregions,whichlackanopenreadingframeand consequentlyhavenoapparentprotein-codingability.Basedon thesize ofthefunctionalRNAmolecule, regulatoryncRNAsare classifiedanalyticallyaslongncRNAs(lncRNAs,>200nt),andsmall ncRNAs(sncRNAs,18–200nt)[1–3].
RNAbiomoleculeshavebeenidentifiedsincethelate1800s,but theirfundamentalrolesincellfunctionshavelongbeen overshad-owedbyDNAandproteins.From1950s,withtheclarificationofthe molecularstructureofDNA,itwasproposedthatRNAwouldbean intermediatemoleculeintheflowofgeneticinformationfromDNA toproteins,asassuredinthecentraldogmaofmolecularbiology. Onthebasisoftheacceptedimportanceofproteinsinexerting bio-logicalfunctions,RNAshavebeenregardedforalongtimeasmerely mediatorsinpassinggeneticcodestofinalproteinmolecules.As aconsequence,thefunctionalactivitiesofRNAsthemselveswere largelyneglected.RibosomeRNAs(rRNA)andtransferRNAs(tRNA) areamongtheearly-discoverednon-codingRNAtranscripts. How-ever,giventheirrolesinfacilitatingproteintranslation,theyare stillconsideredpartofthemachinerytranslatinggenomiccode intoproteinsynthesis.Nonetheless,thediscoveryofthesencRNAs openedthefieldofregulatoryRNAswithnoorlittleprotein-coding potential.Sincethenmanynewclassesofregulatorynon-coding RNAshavebeenidentifiedandremarkableprogresseshavebeen madeinelucidatingtheirexpression,biogenesis,mechanismsand functioninmany,ifnotall,biologicalprocesses,includingcancer developmentandprogression[4,5].
TheideathatRNAsaremuch morethanmoleculesinvolved instorage/transferofinformationemergedsincethediscoveryof ribozymes,endowedwithactiverolesascatalystsofchemical reac-tionsincells.Indeed,RNAhasbeensuggestedtobetheearliest moleculeoflifeandthoughttopossessbothinformationaland cat-alyticfunction[6–8].Thesediscoveriesclearlyencouragedavariety ofstudiestosearchforpotentialnewrolesofRNAmoleculesinvivo, andledtothere-evaluationofRNAsascrucialmoleculesinthe evolutionoflife.Currentadvancesonthesequencingtechnologies revealedthatvastmajorityofthehumangenomeistranscribed, whiletheprotein-codinggenesoccupyonlyabout3%ofthehuman genome[9].Therefore,thewidespreadtranscriptionofthegenome intonon-protein-codingRNAsstronglyimplythatRNAsarecapable ofprocessfunctionsotherthanmeremediatorsbetweenDNAand proteins,andemergingevidencesfromthelasttwodecadeshave unambiguouslyprovedthefunctionalimportanceofnon-coding RNAsin human biology and diseases. As a result, the involve-mentofRNAsinothercriticalmolecularprocessesineukaryotic cellswasprogressivelyrevealed,as inthecase ofDNA replica-tion,proteintranslationandRNAtranscriptmaturation.Withtime, manysmallnon-codingRNA moleculeswereisolatedand char-acterized;amongthefirst,U1,U2,U4,U5andU6smallnuclear RNA(snRNA)asfundamentalpartsofribonucleoproteiccomplexes (RNP),lateridentifiedasthecomponentsofthesplicesome[10]. Thereafter,it wasshowedthat RNAediting mechanisms,based onproteinor protein-RNAcomplexes and regulatingthe infor-mationcontent of tRNA,rRNA and mRNAmolecules, require a “guideRNA”molecule,which,throughbase-pairingwiththetarget RNAmolecule,determinestheeditingsite[11].Inaddition, post-transcriptionalprocessingandmodificationsofrRNAs,essentialfor theproductionofefficientribosomes,isdirectedbytwolargeguide familiesofsmallnucleolarRNAs(snoRNA)[12].
Inthewakeofthesefindings,inthemid-1980sBlackburnand Greidercametothediscoveryoftelomerasebydemonstratingthe existenceofanenzymaticactivitywithincellextractsinserting tan-demhexanucleotidestochromosomeends[13,14].Morerecently itwashypothesizedthattelomerasearisebytheassociationofan ancientribozymewiththereverse-transcriptasesubunit,showing amechanismresemblingthatofpureribozymes,andplacingthe telomeraseasamissinglinkintheevolutionfromRNAenzymesto proteinenzymes[15].
Alreadyatthispoint,thegrowingdescriptionsoftheimportance ofRNAmoleculesforcelllifestartedtopushthemtopublicand scientificinterest,butthecomplexityoftheirrolesandthewide multiplicityofmolecularmechanismsinwhichRNAmoleculesare criticalplayerswasstillfarfromclear.ThediscoveryofmicroRNAs (miRNAs) in early 1990s openeda new chapterof gene regu-lation bynon-coding RNAsand represented a crucial boost for investigationsontheRNAmoleculesnotcodingforproteins[16]. Concurrently,theobservationsthatexogenouslyintroduced dou-blestrandedRNA(dsRNA)andplasmidsexpressingshorthair-pin RNA(shRNA)specificallybase-pairingwithtargetmRNAmolecules wereabletotriggermRNAdegradation(RNAinterference,RNAi) revealed,forthefirsttime,thatspecificsilencingpathwaysbased onsncRNAsareoperatingineukaryoticcells[17,18].These obser-vationsledtothedevelopment oftheRNA interference(RNAi) techniquethathasbeenextensivelyusedinthestudyofgene
func-tion[19,20].
Asmentionedbefore,inrecentyearstheuseofgenomewide approachesandthelargeoutputofgenomesequencing technolo-gieshave revealedthat the mammaliantranscriptome is much morecomplexthanpreviouslyhypothesized,since itincludesa largenumberofsmallnon-codingRNAs(sncRNAs)andlong non-codingRNAs(lncRNAs)[21,22].miRNAsareanabundantclassof endogenousnon-codingsmallRNAmolecules,20÷25nucleotides inlength,whichactaseitheroncogenesortumorsuppressorsgenes and thus have crucial rolesin carcinogenesis[23,24]. Different othertypesofsmallnon-codingRNAshavealsobeensubsequently identified, includingendogenous smallinterfering RNAs (endo-siRNAs), PIWI-associated small RNAs (piRNAs), small nucleolar RNAs(snoRNAs),sno-derivedRNAs(sdRNAs),transcription initia-tionRNAs(tiRNAs),miRNA-offsetRNAs(moRNAs),andothers[25]. Conversely,thelncRNAsfamilycontainsmultipleclassesofRNAs, whicharenuclearRNAstranscriptslongerthan200nucleotides, involvedin theregulationofcellularprocessessuchas apopto-sis,proliferationandmetastasesdevelopment,therebyemerging as important regulators in a wide range of biological activities andhumandiseases[26].Currently,asystematicclassificationof longnon-codingRNAismissing;nevertheless,accordingtotheir genomiclocalizationorotherbiologicalfeatures,theyare classi-fiedasnaturalantisensetranscripts,long intergenicnon-coding RNAs,transcribedultraconservedregions(T-UCRs),circularRNAs, enhancer-associatedRNAs,promoter-associatedRNAs,andothers
[27].SimilartomiRNAs,thedysregulationoflncRNAsisassociated withmanyhumancancersanddefinestheirphenotypes[28].The expressionprofilingofbothlncRNAandmiRNAisdeeplydifferent dependinguponboththehistologicaltypeofthetissuesand patho-logic/notpathologicconditionsastheirexpressionisdifferentin cancertissuesifcomparedtonormalcounterparts.Therefore, lncR-NAsandsncRNAs,bymeansoftheirabilityinpost-transcriptional regulationofgeneexpressionandintargetgenetranslation,may becomeusefulnon-invasivediagnosticbiomarkersandpowerful toolsincancerpreventionandtreatment.Indeed,several emerg-ingevidenceshaverevealedforbothlncRNAsandsncRNAsaclose correlationwithcancerdevelopmentandprogression,sothatsome ncRNAshavealreadybeenusedasbiomarkersandtargetsincancer management,fordiagnosisandtargetedtherapy,respectively[29]. Hence,thediscoveryofthebiologicalfunctionsrelatedtoncRNAs
haveundoubtedlyanimatedthescientificcommunityand stimu-latedbiomedicalstudiesthatarenowadayschangingthewayfor cancerdiagnosisandtreatment.
2. Biogenesisandfunctionalroleofnon-codingRNAs
Theparadoxthatlessthanabout2%ofthetotalhumangenome isrecruitedforproteinexpression,canbepartlyexplainedbythe increase in diversity and functionality of the humanproteome achievedthroughalternativepre-mRNAsplicing,aswellasthrough post-translationalmodificationsofproteins[30].Inrecentyears,it hasincreasinglybecomemoreevidentthatthenonprotein-coding portionofthegenomeisofcriticalfunctionalrelevanceinseveral mechanismsofgeneregulation,bothfornormaldevelopmentand physiology,andforhumandiseases[31].Inparticular,thediscovery that,incomplexorganismsmanytranscribedgenomesequences aredevelopmental-andtissue-regulated,hasstimulated investi-gationsaimedatthecharacterizationofallthedifferenttypesof non-codingregions,originatingnon-codingRNAs[32].
DependingonthencRNAcategory,transcriptioncanbedriven byanyofthethreeRNApolymerases(RNAPolI,II,orIII).Among sncRNAs,shortinterferingRNAs(siRNAs),miRNAsandpiRNAshave beenextensivelystudiedsofarandhavebeenassociatedwith path-waysthatleadultimatelytosilencingofspecificgenesresultingin theprotectionofthecell/genomeagainstviruses,mobilerepetitive DNAsequences,retro-elementsandtransposons[33].
siRNAsand miRNAs(∼20÷30 nucleotides long) derivefrom double-strandedRNA(dsRNA)precursorsthatareintroducedinto cells, or produced endogenously, bygene transcription of both senseandanti-senseDNAstrandsandofpseudogenesandinverted repeats.ThesemoleculesarecriticalinpathwaysengagedinmRNA degradationandtranslationalrepression,therebyregulatinggene expression.Inparticular,miRNAsareconstitutedbyabout22nt andregulatehundredstothousandsofprotein-codingand non-codinggenesbypost-translationalgenesilencing.Itisbelievedthat thehumangenomeencodesthousandsofmiRNAs,butonly1100 havebeendescribedsofar[34].Anumberofevidencehasrevealed up-and/ordown-modulationofmiRNAsinhumanneoplasms, sug-gestingthatmiRNAscanactascanonicaltumorsuppressorsand/or promoters[23–25]. Thus, miRNAexpression profiles havebeen determinedandemployedfordiseaseprognosticationand early diagnosis. Furthermore, most recently, polymorphisms in both miRNAsandtheirbindingsiteshavebeenrelatedto pharmacoge-nomicdifferencesthatcouldexplainchangesin drugactivation andmetabolism[35].Moreindetail,siRNAsaresmallRNAduplex moleculesproducedbytheactionofDicer,aribonucleaseIII(RNase III) enzyme that creates RNA duplexes with2-nt overhangs at their3 ends and phosphate groupsat their5 ends [36]. Con-versely,miRNAsaremostlytranscribedbyRNApolymeraseIIas primary-miRNA(pri-miRNA)moleculeprecursorsequippedwitha characteristicstemloopstructure,andaresubsequentlysubjected toprocessingmechanisms[37].
Inanimals,thefirststepoccursinthenucleuswheretheRNase III Droshaacts over pri-mRNAs (long several hundredsnt still boundtothegeneratingDNA)originatinga pre-miRNA,asmall RNAduplexof∼65÷70nucleotidescontainingthehairpin.This stepcanbeassistedbyRNAprocessingproteins,suchashnRNPA1. Thepre-miRNAsarethenexportedbyanucleartransport recep-torcomplex,exportin-5-RanGTP,tothecytoplasmwheretheyare processedbyDicerinto∼22-ntmaturemiRNAs(miRNA-miRNA* duplexes,wheremiRNAistheantisense,orguide/maturestrand, andmiRNA*isthesense,orpassengerstrand)[38].Thenextstep, forbothsiRNAandmiRNAproduction,isthesubsequent associa-tionwithmembersoftheArgonauteproteinfamilythatdiverge into specialized subfamilies, each recognizingdifferent sncRNA
typesandconferringthespecificfeaturesofthevarioussilencing pathwaysoperatingincells[39].Argonauteloadingoccursinthe RNA-inducedsilencingcomplex(RISC)-loadingcomplex,aternary complexthatconsistsofanArgonauteprotein,Diceranda dsRNA-bindingprotein(knownasTRBPinhumans).Duringloading,the non-guidestrandiscleavedbyanArgonauteprotein.The selec-tionofthedifferentArgonauteproteinsseemstobebasedonthe smallinterferingRNAduplexstructure[40].Thematurationand finalfunctionofcertainmiRNAscanbealsoassociatedto enzy-matic post-transcriptional modifications, like mono-uridylation
[41].ThesemodificationswillincreasethevarietyofmiRNAsand theirprecursor poolsallowingmore complexschemes of regu-lationindifferentbackgrounds.InbothsiRNAsandmiRNAsthe guidestrandsdrivestheRISCstothetargetmRNAsthatcontain complementarysequencesthereby causingtheirdegradationor translationinhibition[42].Ithastobeunderlinedthatinhumans andmammaliansmiRNAsarenotabletoinducedegradationofthe targetmRNAsbuttheycauseonlyitstranslationalrepression.In fact,inmammalstheyassembleonlyattheopenreadingframeat the5ofthemRNAtargetwithpartialcomplementarityinducing thelockofthetranslationofthetarget.Intheloweranimalsandin plants,miRNAscanalsobindtothetargetwithacomplete comple-mentarityinducingitsdegradationbyaRNAsenamedslicer.This modeofactionofmiRNAsinhumanshasanimportanteffect:the amplificationofthebiologicaleffectsinducedbyasinglemiRNA duetotheinhibitionofmultipletargetmRNAs.ThismakesmiRNA potentregulatorsofthecellularfunctionsandpotentialuseful tar-getsincancertherapy[23,24,34].
The piRNA is the largest class of small non-coding RNA molecules expressed in animal cells. The piRNAs (∼24÷31 nucleotides),whose namederives fromthe ability to associate exclusivelytothePIWIsubfamilyoftheArgonauteprotein fam-ily (Piwiproteins),usually have a uridine atthe5 end, holda 5 monophosphate,andpresenta2-O-methyl(2-O-Me) modifi-cationonthenucleotideatthe3 end[43].ComparedtosiRNAs andmiRNAs,widelyexpressedindifferenttissuesandcelltypes, piRNAslackofsequenceconservationandincreasedcomplexity, andhavebeenessentiallydetectedingermcellsofmammals,fish andDrosophilamelanogaster[44].ThisclassofsncRNAshasbeen linkedtobothepigeneticandpost-transcriptionalgenesilencingof retrotransposonsandothergeneticelementsingermlinecells, par-ticularlythoseinspermatogenesis.Indeed,mutationsthatdisrupt thepiRNAbiogenesispathwayarerelatedtogermline-specificcell death,andarealsoassociatedwithincreasedtransposon expres-sion[45].AlthoughpiRNAsbiogenesisandtranscriptionregulation stillneedtobeclarified,itisnowwelldocumentedthattheirorigin differsfromsiRNAsandmiRNAs,becausepiRNAaregeneratedby RNaseIII-independentpathwayswithouttheinvolvementdsRNA precursors.Infact,thesesncRNAsaregeneratedfromlong single-strandedprecursorsthatarepreferentiallycleavedatUresidues andloadedontoPiwiproteins.Inmammals,themajorityof piR-NAsaretranscribedfromdiscretegenomiclocithatareclusteredin largepericentromericorsubtelomericdomains,generallyspanning from100÷100kb,thatcomprisemainlyvarioustransposableDNA elementsandtheirremnants[46].Interestingly,otherstudieshave revealedthat,besidesbeinginvolvedinkeepinggenomeintegrity, asubsetofpiRNAgeneshavebeenimplicatedintheassemblyof thetelomereprotectioncomplex.Recentinvestigationshave fur-thershownthat,inadditiontotheirroleingermlinetransposon regulationandgenomestability,piRNAshaveabroaderfunction inheterochromatinformationanddevelopmentalgeneregulation
[47].
Aspreviouslydiscussed,theotherprominentclassofncRNAs makingupawideportionofthemammaliannon-coding transcrip-tomeisrepresentedbylncRNAs.Thenumber ofgenemembers
integratingthisclassofncRNAsisstillunderdebateandranges from10,000to>200,000.
Differentmechanismsoftranscriptionalregulationof lncRNAs-mediated gene expression have been proposed. Among them, lncRNAsarewell-knowntobeinvolvedinepigeneticDNA mod-ifications through the recruitment of chromatin remodelling complexestospecificloci[48,49].
BiogenesisoflncRNAsisrelativelycomplex.Generally,lncRNA transcriptionandprocessingisextremelycomparableto protein-coding RNAs. Although some lncRNAs could be transcribed by polymeraseIII,themajority oflncRNAsaretranscribed byRNA polymeraseIIfromintergenicregions,promoterregionsor inter-leaved, overlapping or antisense to annotated protein-coding genes [50]. Moreover, there are growing evidences that lncR-NAsmoleculesmightbealsoproduced bytranscriptionalactive pseudogenesand, interestingly,it wasdemonstrated thatsome lncRNAscanbeaswellgeneratedfrommitochondrialgenes[51]. Likecodinggenes,lncRNAsundergopost-transcriptional process-ing, including 5capping, alternative splicing, RNA editing, and polyadenylation.The referred transcriptional origins have been usedtoestablishclassification classesfor lncRNA,as for exam-plepromoter-associatedlongRNAs(lpaRNAs), naturalantisense transcripts(NATs)oropposite-strandtranscripts,largeintervening noncodingRNA(lincRNA),andenhancerassociatedRNAs(eRNA)
[52,53].However,othercriteriashouldprobablybeusedsince
fre-quentlyonelncRNAmoleculecanbeassociatedwithmorethan oneclass.Interestingly,severallncRNAsareantisensetranscripts, alsonamednaturalantisensetranscripts(NATs)[54].NATscanbe classifiedintwosubtypes:cis-NATs,whicharetranscribedfrom oppositeDNAstrandsatthesamegenomiclociandtrans-NATs, whicharetranscribedfromdistalloci[55].Interestingly, numer-ouscancergenes,mainlyofoncosuppressor type, producelong antisensencRNAs,anddespitemRNAandstructuralncRNAs,many lncRNAsaresituatedinthenucleus,someothersarelocalizedin bothcytoplasmandnucleusandothersareexclusivelylocatedin cytoplasm[56].
3. Mechanismandclinicalrelevanceofexosome-mediated
miRNAsecretion
About15yearsafterthefirstidentificationofmiRNAs,ithas beendiscoveredthattheyweredetectableinbodyfluids encap-sulated in lipid microvesicles [57]. Extracellular vesicles (EVs), characterized according to the size into exosomes (<100nm), microvesicles(1000nm)andapoptoticbodies(1–4m)[58], orig-inatefromcellsandareableoftransferringmiRNAs,mRNAsand proteinsinbothparacrine(connectingcellsbelongingtothesame tissue)andendocrine(todistanttargetcells)manner[58].In addi-tiontoEV,circulatingmiRNAscanbealsoloadedintohigh-density lipoprotein(HDL)[59,60],orboundinaproteincomplexcomposed ofArgonaute(AGO)familymembersoutsideofvesicles[61]. Col-lectively,eachofthesemechanismsisabletoprotectmiRNAsfrom degradationensuringtheirstability,sincethenakedRNAwouldbe readilytargetedbytheendonucleasesthatareabundantlypresent inextracellularfluids.Amongthesemechanisms,exosomeshave beendefinedascrucialmediatorsoftheintercellular communica-tionandpromisingvehiclesformiRNAdeliveryandgenetherapy. Infact,accumulatingevidencesuggestthatmiRNAsplayan impor-tantroleinexchanginginformationbetweencellsand,therefore, theymayserveasnewandpotentiallyusefulmarkersand pow-erfultargetsfortherapeuticinterventionsagainstvarioushuman diseases.Themostabundantcomponentsofexosomemembranes arerepresentedbylipidsandproteins[62–64],giventheir deriva-tionfromplasmamembrane. Asshownin Fig.1,exosomesare firstlyproducedbyendocytosis,whenthecellularmembrane is
internalizedtoproduceendosomes;thereafter,theinvaginationof endosomemembranesoriginatesmanysmallvescicleswithinthe sameendosome,whichisthusnamedmultivescicularbody(MVB). MVBsarethenreleasedfromthecellintotheextracellularspace byfusingwiththecellmembrane,leadingtotheformationof exo-somes[65].Allthesestepsofformationandsecretionrequirethe involvementofenzymes[66,67]andATP[68].Withinthesetof regulatorymoleculesimplicatedinthesecretionphase,thereare Rab27aandRab27b,whoseknockdownhasbeencorrelatedwith theinhibitionofthisprocessinHeLacells[69].Furthermore,the oncosupressorproteinp53anditsdownstreameffectorTSAP6are alsoinvolved inthisregulatorynetworkbeingabletostimulate exosomeproduction[70].
ConcerningmiRNAsortingintoexosomes,currentliteraturehas describedatleastfourpotentialmechanisms.Thefirstmodeof sort-ingtobediscoveredisbasedontheneuralsphingomyelinase2 (nSMase2)-dependentpathway;overexpressionofnSMase2was, infact, putincorrelation withtheincreasednumberof exoso-malmiRNAsand,ontheotherhand,theinhibitionof nSMase2 wasassociatedwiththereductionofexosomalmiRNAlevels[67]. Thesecondmechanisms,discoveredbyVillarroya-Beltrietal.,view theinvolvement of theGGAG miRNA motif (in the30 portion ofmiRNAsequence)thatcanberecognizedbysumoylated het-erogeneousnuclearribonucleoproteins(hnRNPs)responsiblefor thespecificpackagingofmiRNAsintoexosomes[71].The30-end miRNAsequence-dependentpathwayisthethirdmodeofsorting, describedbyKoppers-Lalicetal.;indetail,30endsofuridylated miRNAsweremostlyrecognizedinexosomesderivedfromBcells or urine, whereas the30 ends of adenylated endogenous miR-NAswereprincipallyfoundinBcells[72].Thefourthmechanism involvesthemiRNAinducedsilencingcomplex(miRISC).Indetail, miRISCsco-localizewiththesitesofexosomebiogenesis(MVBs) andtheircomponents,suchasAGO2protein(whichpreferentially bindstoUorAatthe50endofmiRNAs)andmRNAtarget,arealso correlatedwithsortingofmiRNAsintoexosomes[73].
Exosomespresentinbodyfluidscaninteractwithrecipientcells throughthreemainmechanisms:i)thedirectinteractionbetween transmembraneproteinsofexosomesandthesignalingreceptors oftargetcells[74];ii)theexosomesfusionwiththecellular mem-braneofrecipientcellswiththefollowingdeliverytothecytosol
[75];iii)theinternalizationofexosomeswithintherecipientcells. Subsequently,the fateofexosomesmay followtwo alternative routes:i)fusionwithendosomesandtranscytosis,withfollowing releaseofthecontent,representedbymiRNA,withintherecipient cellor,otherwise, ii)maturationofendosomes,fusedwith exo-somes,intolysosomesandsubsequentdegradationoftheircontent
[75,76].Globally,theuptakeofthislipidvesciclesbyrecipientcells
seemstoinvolve clathrin-and caveolin-dependentmechanisms
[77,78].Anyhow,ithasbeenproventhatdisruptionofexosomal
lipidraftscounteracts theinternalizationofexosomesand that annexins,which areassociated withcell-to-celladhesion,were crucialforexosomesuptakeinbreastcancercells[79].Moreover, inovariancancercells,treatmentwithproteaseKdetermineda cleardecreaseofexosomeuptake, thusconfirmingthe involve-mentofsurfaceproteinsintheinternalizationofexosomes[80]. However,thedetailedmechanismofexosomeinternalizationstill needsfurtherclarification.
Clinicalapplicationofexosomesdetectableincirculation con-sists in the analysis of their cargo, expressly represented by miRNAs,whichmayspecificallyreflectthepathogenesisofthecell oforiginthatcanbetransferredtorecipient cellsaltering their molecularcharacteristicswithinevitableeffectsoncellfunction. CirculatingmiRNAs stabilized in exosomes are,therefore, ideal candidatesfortheidentificationofnon-invasivecancer biomark-ersdetectableinbodyfluidssuchasserum,blood,saliva,urine, tearfluidandbreastmilk[81,82].Aclearadvantageinemploying
Fig.1. ExosomeformationandmiRNAsorting.
Exosomesarefirstlyproducedbyendocytosis,whenthecellularmembraneisinternalizedtoproduceendosomes;thereafter,theinvaginationofendosomemembranes originatesmanysmallvescicleswithinthesameendosome,whichisthusnamedmultivescicularbody(MVB).MVBsarethenreleasedfromthecellintotheextracellular spacebyfusingwiththecellmembrane,leadingtotheformationofexosomes.AllthesestepsofformationandsecretionrequiretheinvolvementofproteinsandATP. miRNAsortingintoexosomescanfollowatleastfourpotentialmechanisms:1)basedontheneuralsphingomyelinase2(nSMase2)-dependentpathway;2)bysumoylated heterogeneousnuclearribonucleoproteins(hnRNPs);3)basedontherecognizionofthe30-endofuridylatedmiRNA;4)bymiRNAinducedsilencingcomplex(miRISC). Exosomesreleasedinbodyfluidscaninteractwithrecipientcellsthroughthreemainmechanisms:thedirectinteractionbetweentransmembraneproteinsofexosomes andthesignalingreceptorsoftargetcells;theexosomesfusionwiththecellularmembraneofrecipientcellswiththefollowingdeliverytothecytosol;theinternalization ofexosomeswithintherecipientcells.
exosomalmiRNAsinsteadofAGO-miRNAsasbiomarkersis repre-sentedbytheirstabilityduringtheprocessingofgeneticmaterial incirculation.Infact,theseprocessescauseanextensivehemolysis thatmayinducethereleaseofadditionalAGO-miRNAsfromcells withsubsequentcontaminationofbloodwithirrelevantmiRNAs, thuscomplicatingtheanalysis.Instead,theseissuesarereduced whentheanalysisisperformedonmiRNAinternalizedinexosomes
[83].SeveralexosomalmiRNAshavebeenidentifiedasbiomarkers forcancer,provingtheirclinicalutilityandtheirpotentialinthe fieldofpersonalizedmedicineanddiagnostics.Particularly, exo-somal miRNAshave severalfunctionsrelated withcell growth, migration, invasion,metastasisand impairment of theimmune systemresponse[84].Insomecasesithasbeenidentifieda cell-independentmiRNA biogenesis,associatedwithexosomes,able toalterthetranscriptomeofnon-cancercellstransformingthem incancercellsinaDicer-dependentmechanism[85].Theprocess ofmetastaticcascadeactivationduringtumorprogressioncanbe coordinatedbyexosome-mediatedsecretionofmiRNAs. Particu-larly,itwasobservedanaugmentedamountoftheoncosuppressor miRNAs,suchasmiR-23b,secretedfrommetastaticcellsderived frombladdercancer,suggestingthatthereleasefromexosomes maycontroltheamountofintracellularmiRNAsindonorcellsand affectthelevelsofthesamemiRNAsinrecipientcells[86].Basedon thecentralroleofexosomalmiRNAsincell-tocellcommunication, theycoulddrivethedevelopmentofanovelclinicalapplication foundedontheemployofanewclassoftarget-basedanticancer agentsdirectedversusmiRNApathways.Alongsidetherestoring
of oncosuppressor miRNAs with syntheticmiRNA mimics – an emblematiccaseisrepresentedbymiR-34athatisthefirst cancer-targetedmiRNAdrug(MRX34)enteringPhaseIclinicaltrials[87]
–andtheblockageofoncogenicmiRNAswithantisense oligonu-cleotides,anothertherapeuticapplicationmightbetheblockage ofthesecretionoftumorcell-derivedexosomes.Nevertheless,this procedurecouldnotberecommendable,sinceexosomesincludea broadrangeofothermoleculesasidefromoncogenicmiRNAs.For thatreason,apossiblestrategycouldconsistinthetherapeuticuse ofcellsthatpreferentiallysecreteaspecificcategoryofexosomal miRNA,suchasmesenchymalstemcells(MSCs),whosesecreted miRNAsaremainlyinvolvedintissuerepair[88].Moreover,the relativeabundanceofcirculatingmiRNAswithpredominanttumor suppressoractivityinthebloodofhealthyindividuals,actingasa surveillancemechanism,andthespecificalterationofmiRNA sig-natureincancerpatientsrepresentsanencouragingperspective inviewofidentifyingstableandreliablebiomarkers.Overall,the current scientificreports analyzingtheexosomalmiRNAsagree ontheirpromisinguseforvariousclinicalapplications,including treatmentanddiagnosisofneoplasms,althoughitisclearthatitis necessaryanintensiveinvestigationofexosomalmiRNAsbiology.
4. ncRNAdysregulationincancer:preclinicalstudiesand
therapeuticimplications
RecentstudiesindicatethatmiRNAsarederegulatedinmany typesoftumors,beinginvolvedinseveralcancerprocesses,such
ascellproliferation,invasionandmetastasis.miRNAscan,infact, inhibittheexpressionof genesinvolved inmany cellular path-waysregulatingcrucialmechanismslikecellcycle,celldeathand cellmigration. Numerousexperiments and clinic analyses sug-gest that miRNAs may function as a novelclass of oncogenes (oncomirs) or tumor suppressor genes. Oncomirs are overex-pressedintumorsandactbypromotingtumordevelopmentand bynegativelyinhibitingonco-suppressivegenesand/orgenesthat controlcelldifferentiationandcelldeath.Agoodexampleforan oncogenic miRNA is miR-21. It is overexpressed in most types ofmalignancies,includingbreastcancer,glioblastoma,colorectal cancer,lungcancer,pancreaticcancer,andleukemia[89–91]. Func-tionalstudiesincancercelllines indicatethatmiR-21 playsan importantroleintheoncogenicprocessasindicatedbyits asso-ciationwithhighproliferation,lowapoptosis,highinvasion,and metastaticpotential[92–94]. Anumber of miR-21 targetgenes havebeen identified, includingPTEN, PDCD4, and BTG2, which playimportantrolesintheoncogenesis[95].Inglioblastoma, miR-21wasrevealedtotargetseveralimportantcomponentsofthe epidermal growth factor receptor (EGFR) and phosphatase and tensinhomolog(PTEN)signalingpathways.InhibitionofmiR-21by specificantisenseoligonucleotidesinU251MGglioblastomacells decreasedtheexpressionofEGFRandactivatedAKT,CYCLIND,and
BCL2[96,97].AnotherwellknownoncosuppressormiRNAis
miR-34a,whoseenforcedexpressioninmultiplemyeloma(MM)cells andinMMmousemodels,inducessequentialinhibitionofErkand Aktactivitiesandcaspase-3and-6cleavage,aswellasBcl-2and NOTCH1downregulation[98].PreclinicalmodelsofMMhavealso allowedtoidentifyatumorsuppressorrolealsoformiR-29b.Itacts asepigeneticregulatorthroughtheinhibitionofHDAC4,whichis highlyexpressedinMMand,therefore,representsarelevant tar-getfortherapy[99].AnotheroncogenictargetofmiR-29bisthe transcriptionfactorSp1,whichhasbeendemonstratedtobe,in turn,anegativeregulatorofmiR-29b,thusestablishinganegative feedbackloop[100].
During tumorigenesis, the expression of some miRNAs is decreased,sothesetypesofmiRNAsareconsideredtumor sup-pressorgenes.TumorsuppressormiRNAsnegativelyinterferewith tumordevelopmentinhibitingoncogenesand/orgenesthatcontrol celldifferentiationorapoptosis.AmiRNAconsideredastumor sup-pressorgeneislet-7.Itplaysapivotalroleintumorsuppressionin manycancers,includingesophagealsquamouscellcarcinoma,lung cancer,nasopharyngealcarcinoma,andprostatecancer[101–104]. Takamizawaetal.[105]foundthatlet-7waspoorlyexpressedin lungcancers;moreover,reducedlet-7expressionwassignificantly associatedwithshortenedpost-operativesurvivalindependentof diseasestage.Thissuggestedthatlet-7maybeatumor suppres-sorgene.Toconfirmthisconclusion, theauthorsoverexpressed let-7geneinA549lungadenocarcinomacelllinesandinducedthe inhibitionoflungcancercellgrowthinvitro[105].
InadditiontomiRNAs,lncRNAshavealsoemergedas impor-tantregulatorsinbothoncogenicandtumorsuppressorpathways
[106].Somestudiesallowedtheanalysisof ncRNArolein
can-cer,evaluatingtheexpressionofmorethan10,000lncRNAgenes inmorethan1000tumorsamples.In thesestudiestheauthors identifiedthelncRNAsassociatedwithspecifictumorsandtheir role in oncogeniccell growth [107]. Otherstudies have shown thatsomelnRNAsaremoreexpressedandmoreactiveintumors thaninnormalcells[108–110].Accumulatingevidenceprovides mechanisticinsightdemonstratinghowlncRNAsregulate impor-tantcellularsignalingpathwaysincancercellsattranscriptional, post-transcriptional,andepigeneticlevels[111].Inarecentstudy, theauthorsanalyzedtheexpressionandfunctionofPCAT-14in hepatocellularcarcinomaHCC.LncRNAprostatecancer-associated transcripts(PCATs) wereoriginallyidentified as biomarkers for prostatecancer[112].Inparticularinthisstudytheauthorsshowed
thatthelncRNAPCAT-14isoverexpressedinpatientswithHCC,and isassociatedwithapoorprognosisaftersurgery.PCAT-14promotes proliferation,invasion,andcellcyclearrestinHCCcellsandinhibits miR-372expressionbyinducingmethylationofthemiR-372 pro-moter [113]. Other lncRNAs have been associated with tumor suppressorfunctions.Inliteratureitisdemonstratedthatseveral lncRNAsaredirecttranscriptionaltargetsofp53,andknockdown ofspecificlncRNAsmodulatesp53-inducedapoptosis.Indetail,a studyrevealedthatthelncRNANEAT1isadirecttranscriptional targetofp53.ThesuppressionofNEAT1inductionbyp53 atten-uatestheinhibitoryeffectofp53oncancercellgrowthandalso modulatesgenetransactivation,includingthatofmanylncRNAs. Furthermore,lowexpressionofNEAT1isrelatedtopoor progno-sisinseveralcancers.Theseresultsindicatethattheinductionof NEAT1expressioncontributestothetumor-suppressorfunctionof p53andsuggestthatp53andNEAT1formatranscriptional net-workcontributingtovariousbiologicalfunctionsincludingtumor suppression[114].
OtherncRNAspeciessuchaspiRNAsandsnoRNAsare obtain-ingagreaterappreciationfortheirroleincarcinogenesis[115].In detail,piRNA-651wasfoundtobeup-regulatedinseveralcancer celllinesincludinggastric,lung,mesothelial,breast,liver,and cer-vicalcancercells[116];moreover,itwasreportedalsoforhuman snoRNAsanimportantroleintumorigenesis[117].
InthelightofthisevidenceitcanbestatedthatncRNAshave beenrecognizedaspromising therapeutictargetsforanticancer treatments.Inparticulara miRNAcansimultaneouslymodulate itsmultipletargetgenes,alteringoncogenicandtumorsuppressor pathways.Cancer-associatedupregulationofoncogenicmiRNAs, canbecounteractedbyseveralmeans.Basedontheevidencethat miRNAscontroltheirtargetsthroughbasepaircomplementarity, antisenseoligonucleotides(ASOs)havebeendevelopedtoinhibit theirfunction.InordertoincreaseASOs’stabilityandefficacy, dif-ferentchemicalmodifications,suchaslockednucleicacids(LNAs), anti-miRNAoligonucleotides (AMOs),andantagomirs,are intro-duced.Forexample,aspecificantagomirwasusedtoknockdown theoncogenemiR-21inbreastcancerMCF-7cells,resultingin sig-nificantinhibitionofMCF-7growthinvitroandintumorxenografts throughinhibitingcellproliferationandinducingapoptosis[118]. AsfarasitisconcernedtumorsuppressormiRNAs,miRNAmimics orlentiviralvectorscanbeusedtorestoretheirexpressionlevels. Therequestoflongcirculationinthebloodandofpreferential accumulationinthetargetsiteasasafetyrequirementhas stim-ulatedtheexplorationofnonviralvectors,suchasnanocarriers. Nanocarriersaresmallparticles(rangingfrom1to300nm)that cancarryanddeliverdrugs,oligonucleotides,peptidesordesired cargostotargettissues.Variousnanocarriershavebeenusedfor ncRNAdeliveryinbiomedicalapplications.Basedonsurfacecharge, sizeandhydrophobicity,theyhaveuniquetissuebiodistribution, toxicity,andtumorcelluptakeprofiles[119].Recently,ithasbeen reportedthatdirectconjugationofsmalldrugmoleculesto ncR-NAscanimprovetheinvivopharmacokineticbehaviorofncRNAs. LncRNAsrepresentanimportantresourceintermsofdeveloping diagnostics andtherapies becausemany of themare expressed ina tissue-andcancer-type specificmannerandcouldbecame novelbiomarkers.ThediscoverythatlncRNAcouldbedetectedin thebody fluidofcancerpatientsopenedupa newandexciting possibilityofusinglncRNAsasnon-invasivebiomarkers.The devel-opmentofmoreefficientandspecificdeliverysystemisnecessary toachievehightherapeuticefficiencyandtargetspecificity.Also forthelncRNAstherearedeliverysystemstargetingtherapeutics: viraldeliveryandnon-viraldelivery.Viraldeliverygenerally pro-videshighgenetransferefficiency,butisdeficientinbiosafetyand, therefore,notappropriateforhumanusedespitethepromising resultsinanimalmodels.Currently,numeroussyntheticdelivery systemshavebeendevelopedtomanipulatethegenetransfer
effi-ciency,andsomeofthemhavedemonstratedpromisingresultson humans[120,121].
5. PotentialofncRNAsasnovelbiomarkersforsolidand
hematologicmalignancies
ImprovedknowledgeofncRNAs’expressionpatternand func-tionmayleadtoabetterunderstandingoftheheterogeneityof malignanciesand,mostlikely,alsoleadtotheiruseas diagnos-tic,prognosticandtherapeutictargets.Thebest-studiedncRNAs categoryisrepresentedbymiRNAs,whichhaveemergedas suit-ablediagnosticand prognosticbiomarkers withthecapacityto drivetreatmentdecisionsintheclinicalsetting.Researchershave identifiedmiRNA signaturesin serum,plasma,peripheralblood mononuclear cells and whole blood, giving theopportunity to distinguishpatientswithdifferentcancers,suchasprostate (miR-141),lung(miR-21,miR-210andmiR-486-5p),breast(miR-195 andlet-7a)andlymphoma,fromhealthyindividuals[122].Some ofthemostderegulatedmiRNAsarelistedinTable1.
5.1. miR155
Emergingevidence suggestthat miR-155is significantly up-regulatedinlung cancertissues,plasmaandsputum,and could serve as a promising marker for the diagnosis and poor prog-nosisofnon-small celllungcancer(NSCLC)[123].On theother hand,overexpressionofmiR-155inhumanbreastcancercellshas shownaprotectiveroleintriple-negativebreastcancersthrough RAD51targeting,alsoaffectingthecellularresponsetoionizing radiation.Furthermore,highmiR-155levelswereassociatedwith lowerRAD51expressionandwithbetteroverallsurvivalofpatients in a largeseriesof triple-negative breast cancers [124]. A high numberofstudieshaveinvestigateditspotentialasabiomarker inseveralB-cellmalignancies,but conflictingresultshavebeen presented. Costinean et al., demonstrated that transgenicmice over-expressingmiR-155inB-cells,developedapre-leukemic pre-B-cellproliferation,followedbyaB-cellmalignancy[125].Astudy ofmiR-155KOmiceshowedthatmiR-155regulatesgerminal cen-terreactionandT-helpercelldifferentiationbyaffectingcytokine production,andthatBIC/miR-155regulatesthefunctionofboth lymphocytes and dendritic cells leading to defective immune response[126].Therefore,inmicemodels,miR-155deregulation in Bcells causes acutelymphoblastic leukemiaand high-grade lymphomas[126],whereasinmyeloidcellsitisassociatedwith myeloproliferative disorders [127,128]. In addition, high levels ofmiR-155werealsofoundinnewlydiagnosedcytogenetically normal acute myeloid leukemia bearing FLT3-internal tandem duplications,associatedwithpoorprognosis[129].
5.2. miR-29
ThemiR-29 family comprises of three membersin humans, miR-29a,miR-29bandmiR-29c,differentlyexpressedin several solidorhematologictumors,suchas,nasopharyngealcarcinoma
[130],non-smallcelllungcancer[131],hepatocellularcarcinoma
[132],breastcancer[133],cutaneousmelanoma[134]anddiffuse
largeBcelllymphoma[135].Performingasystematicreviewofthe literaturewithameta-analysisaimedtoevaluatetheprognostic valueofthemiR-29familyexpressionindifferenttypesof can-cers,Qietal.foundthatthelowexpressionofmiR-29isassociated withaggressivenessandpoorprognosisofmalignantneoplasms
[136].In 2005,Calinetal.[137]reportedforthefirsttimethat
the miR-29 family could discriminate good or poor prognosis between chronic lymphocytic leukemia samples. Also in man-tlecelllymphomamiR-29wasidentifiedasaprognosticmarker and pathogenic factor that targeted cyclin-dependent kinase 6
(CDK6) [138]. Moreover, Garzon et al. [139] reported that the expressionofmiR-29familywasdownregulatedinacutemyeloid leukemia(AML)patientswitht(11q23)chromosome rearrange-mentsvsallotherAMLpatients,althoughmiR-29familyexpression wasupregulated inAMLwithpositivecytoplasmic nucleophos-min(NPMc+AML)[140].Recently,severalstudieshaveidentified miR-29asanon-invasivediagnosticandprognostictoolfor col-orectalcancer(CRC).SerumlevelsofmiR-29bandmiR-194were foundtobesignificantlylowerinCRCpatientsascomparedwith controlsubjects.Furthermore,serumlevelsofthismiRNA were inverselycorrelated withtheadvancedtumour-node-metastasis (TNM)stages[141].Inoueetal.evaluatedtheassociationbetween miR-29bexpressionandsurvivalin245patientswithCRC.They noticedlowermiR-29blevelsinserumofCRCpatients,alsofinding furtherdecreasedexpressioninadvancedclinicalstagesoftumor
[142].RegardingmiR-29a,upregulatedexpressionwasdetectedin
serumfromcolorectallivermetastastaticpatientscomparedwith non-metastasizedCRCpatients[143].Moreover,reduced expres-sionofmiR-29a,withmiR-223andmiR-224,wasfoundinthefeces fromtheCRCpatients,thusprovidinginformativebiomarkersfor bothscreeningandearlydiagnosisofCRC[144].
5.3. miR-17-92family
TheoncogenicmiR-17-92familyiscomposedof3related poly-cistronicmiRNAgeneclusters:miR-17-92clusteranditsparalogs miR-106b-25 and miR-106a-363clusters [145].The miR-17-92 cluster,transcriptionallyactivatedbyc-MycandE2F,isupregulated inchroniclymphocyticleukemiacellsandinvariouslymphoma celllines [146].In mantlecell lymphoma,upregulationof miR-17-92antagonizeschemotherapy-inducedapoptosisbyinhibiting proteinphosphatase PHLPP2,anadditional targetofmiR-17-92 and an important negative regulator of the PI3K/AKTpathway
[147].ThefindingthatmiR-17-92expressioncontributestothe
signatureofhematologictumorcelllinesprovidesevidencethat miRNAexpressionpatternsreflectthepatternsobservedinprimary hematologicmalignancies[148].miR-17-92clusterhasalsobeen demonstratedtoplayacrucialroleinvariousotherhumancancers. Highexpressionlevelshavebeenfoundinosteosarcomatissues witharelationshipbetweenmiR-17-92clusterupregulationand advanced TNMstage,aswellaspoorer recurrence-freesurvival ofosteosarcomapatients.[148].Moreover,miR-17-92clusterhas beenidentifiedaspotentialserumbiomarkerfortheearlydetection ofbothgastriccancerandintestinalmetaplasia[150].
Along withmiRNAs, alsolncRNAs couldact asnon-invasive tumor markers in both diagnosis and prognosis prediction
[151,152].LncRNAshavebeenshowntoregulatecellularprocesses
thatarepertinenttocancerdevelopment,includingcellcycle pro-gression[153],apoptosis[154]andmetastasis[155](Table2). 5.4. NEAT1
Nuclear enriched abundant transcript 1 (NEAT1) is a novel lncRNA specificallylocalizedto nuclearparaspeckles[156]that seemstobeinvolvedinregulatinggeneexpressionbyretaining mRNAsforeditinginthenucleus[157].InNSCLC,NEAT1 expres-sionwasrelatedtopatientage,vascularinvasionandclinicalTNM staging[158].AnotherstudyshowedthatcirculatingNEAT1levels weresignificantly higher in NSCLCpatients’ plasma [159], sug-gestingthatincreasedNEAT1expressionmightbeassociatedwith progressionofNSCLC,wherecirculatingNEAT1couldbeusedas a diagnosticmarker.In addition, NEAT1couldinduce hsa-miR-377-3ptoderepressE2F3,whichisakeyoncogeneinpromoting NSCLC[160].Inlaryngealsquamouscellcarcinoma(LSCC),NEAT1 overexpressionwasassociatedwithadvancedclinicalstageand lymphnodemetastasis.Moreover,Wangetal.,showedthatNEAT1
Table1
miRNAderegulationincancerdiseases.
miRNA DiseaseAssociation Expression Role BiomarkerApplication References
miR-155
Non-smallcelllungcancer upregulated Oncogene Diagnosisandpoorprognosis [123–129]
Breastcancer Oncosuppressor Predictsbettersurvival
B-cellmalignancy Oncogene Myeloproliferativedisorders
Acutemyeloidleukemia Oncogene Predictspoorprognosis
miR-29
Nasopharyngealcarcinoma downregulated Oncosuppressor Aggressivenessandpoorprognosis [130–144] Non-smallcelllungcancer
Hepatocellularcarcinoma Breastcancer
Cutaneousmelanoma Prognosticmarker
Bcelllymphoma
Chroniclymphocyticleukemia Diagnosisandmonitoring
Acutemyeloidleukemia Colorectalcancer
miR-17-92
Chroniclymphocyticleukemiacells upregulated Oncogene Riskfactor [145–155]
Mantlecelllymphoma Predictstherapeuticresponsiveness
Osteosarcoma PredictsadvancedTNMstageaand
poorerRecurrence-freesurvival.
Gastriccancer Potentialserumbiomarkerforthe
earlydetection Intestinalmetaplasia
Table2
lncRNAderegulationincancerdiseases.
LncRNA DiseaseAssociation Expression Role BiomarkerApplication References
Neat-1
Non-smallcelllungcancer upregulated Oncogene Prognosisandclinical
tumour-node-metastasisstaging.
[156–170] Laryngealsquamouscellcarcinoma Poordifferentiation,metastasis,invasion
Colorectalcancer,Hepatocellular carcinoma,Breastcancer,Oesophageal squamouscell
Prostatecancer Predictstherapeuticresponsiveness
Ovariancarcinoma Diagnosis
Glioma Shortenedoverallsurvivalandtumour
recurrence.
Acutemyeloidleukemia Potentialtherapeuticuse
Malat-1
Non-smallcelllungcancer upregulated Oncogene Metastasesandprognosisinearlystage. [171–180] Predictstherapeuticresponsiveness.
Osteosarcoma Recurrenceafterlivertransplantation.
Hepatocellularcarcinoma
ClearcellrenalcellcarcinomaGlioma Tumorprogressionandpoorprognosis.
Pancreaticcancer Diagnosisandmonitoring
Colorectalcancer Multiplemyeloma
Hotair
Breastcancer upregulated Oncogene Prognosticvalueformetastasisand survival
[181–186]
Hepatocellularcarcinoma Stagemarkerandhistological
differentiation
OralsquamouscellcarcinomaB Pooroverallsurvivalandrelapse-free survivaltimes
Acutemyeloidleukemia
Cmultirow20.5inDLEU1/2 Chroniclymphocyticleukemia downregulated Oncosuppressor Predictspoorprognosis [187,188] B-celllymphomasMultiplemyeloma
regulatesCDK6expressionthroughmodulatingmiR-107andits knockdowninducestheapoptosisofLSCCcellsinvivo[161].NEAT1 expressionwasassociatedwithpoordifferentiation,metastasis, invasion,aswellasTNMstagingincolorectalcancer[162], hep-atocellularcarcinoma[163],breast cancer[164]andesophageal squamouscellcarcinoma[165].Inprostatecanceritinduces resis-tancetoandrogenreceptorantagonists[166].Inaddition,NEAT1 wassignificantlyupregulatedinstageIIIserousovariancarcinoma
[167]. Pils and colleagues performed genome-wide expression
analysisinleucocytesisolatedfrom44epithelialovariancancer patientsand 19 normalcontrols, showing that NEAT1, in com-bination of 12 remaining genes and 6 plasma proteins, might discriminatepatientswithepithelialovariancancerfromnormal controlswithasensitivityof95.6%andaspecificityof99.6%[168]. Inglioma,highNEAT1expressionwasalsoindependently asso-ciatedwithshortenedoverallsurvivaland tumorrecurrence,as
wellaslargertumorsizeandWHOgrade.Moreover,highNEAT1 expressioninpatientswithstageIII–IVgliomaindicatedpoor prog-nosis[169].NEAT1expressionlevelissignificantlyrepressedby PML-RAR␣oncoproteinindenovoacutepromyelocyticleukemia (APL).When all-trans retinoic acid(ATRA) has been used as a leukemiatherapytotargetthetranscriptionalrepressionmediated bythePML-RAR␣fusionprotein,asignificantNEAT1upregulation wasobserved,inparallelwithATRA-inducedNB4cell differentia-tion;thiseffectwasblockedbyNEAT1inhibition.Thesefindings indicatedthataberrantNEAT1downregulationcontributestothe blockadeofdifferentiationinAPL[170].
5.5. MALAT-1
MALAT-1 (metastasis associated lung adenocarcinoma tran-script1)isanintergenictranscript(7kb)locatedonchromosome
11[171]implicatedinseveralmalignancies.Itwasfirstlycorrelated withmetastasesandprognosisinearlystageNSCLC[172]and sub-sequentlywithchemotherapeuticresponseinosteosarcoma[173]
andrecurrenceofhepatocellularcarcinomainpatientsundergoing livertransplantation[174].Itissignificantlyoverexpressedinmany typesofsolidcancersinwhichitcorrelateswithtumorprogression andpoorprognosis:inclearcellrenalcellcarcinoma[175]through theinteractionwithEzh2andmiR-205[176],inglioma[177]and pancreaticcancer[178],inCRCthroughthebindingtoSFPQand thereleasingofPTBP2oncogenefromSFPQ/PTBP2complex[179]. MALAT-1isalsooverexpressedinhematologicmalignanciesasin MMandmayserveasamarkertopredictdiseaseprogression[180]. 5.6. HOTAIR
HOXtranscriptantisenseRNA(HOTAIR)isa2.2kb,long inter-genic non-coding RNA (lincRNA) localized to the HOXC locus (12q13.3)[181].Itfunctionsasamolecularscaffoldtolinkthe poly-combrepressivecomplexes2andthelysinespecificdemethylase1 complexes,andregulatesgeneexpressionbymediatingthe mod-ulationofchromatinstructuresintransacrossthe40-kbHOXD locus[182],promotingcancermetastasis[183].HOTAIRis system-aticallydysregulatedinbreastcancer[183]andinhepatocellular carcinoma[184],andit hasprognosticvalueformetastasisand survival.HOTAIRexpressionincreasedalsoinoralsquamouscell carcinoma(OSCC),whereitisassociated withmetastasis,stage, histologicaldifferentiation,pooroverallsurvivalandpoor disease-freesurvivalinOSCCpatients.Furthermore,thislncRNApromotes epithelial-mesenchymaltransition(EMT)byrepressingE-cadherin expressionthroughtherecruitmentofEZH2andH3K27me3[185]. Moreover,HOTAIRmayrepresentabiomarkerforpooroverall sur-vivalandpoorrelapse-freesurvivalinAMLpatientswithhigher HOTAIR expression levels, withrespect to patients withlesser expressionlevelsofthelncRNA[186].
5.7. DLEU1/DLEU2
Deletedinleukemia1(DLEU1)and2(DLEU2)genesare tran-scribedina30-kbregionsituatedinthelongarmofchrormosome 13(13q14),whichismissinginmorethan50%ofpatientswith chroniclymphocyticleukemia(CLL)andcanbeassociated with thepredictionofpoorprognosis[187]inthisneoplasm,aswell asinotherB-cellmalignancies,includingdenovoandtransformed diffuselargeB-celllymphomas,andMM[188].Ithasbeen demon-stratedthatintron4ofDLEU2encodesformiR-15aandmiR-16-1 clusterthat,aspreviouslyreported,canexertacrucialroleinthe tumorigenesisofCLL,inpartthroughtheregulationoftheoncogene
BCL2[187].
6. miRNAdetectionandquantificationinbodyfluids
miRNAs,unlike other biomarkers,are highly stable and can beisolatedfromtumortissuesamplesafterformalinand paraf-finpassages,andfromserumandplasmasamplesafterbeingat roomtemperaturefor24handafterfreezingandde-freezingcycles
[189].Inaddition,miRNAsappeartoberesistanttoRNAsespresent
intheplasmaprobablyduetotheirsmallsizeormolecular struc-ture,aswellasinvirtueoftheirloadinginEV.Thepresenceof circulatingmiRNAshasbeendemonstrated inseveralbiological samples suchas serum, tears, urine, amnioticand ascitic fluid
[190]. The correlationbetween differentprofiles in body fluids
andthetumorprogression,thepresenceofmetastasesand, conse-quently,theprognosis,makeitpossibletousethesemoleculesas biomarkers.TheexpressionprofileofcirculatingmiRNAsishighly influencedbysamplepreparationmethods[191,192].The extrac-tionofRNAisusuallyperformedbyusingcommerciallyavailable
techniquesincludingphenol/guanidiniumproducts,suchasTRIzol (LifeTechnologies),andcolumn-basedextractionkits,suchas mir-Vana(LifeTechnologies)andmiRNeasy(QIAGEN).Inmoststudies, theextractedmiRNAsaresubjectedtonext-generationsequencing (NGS)ormiRNAmicroarraystoobtainlarge-scaleprofilesof cir-culatingmiRNAsandtodeterminecandidatemiRNAsforfurther quantification.Thesemethodshavebothadvantagesand limita-tions.NGShasapotentialtoidentifynovelmiRNAsbutitisless cost-effectiveandless efficientcomparedwithmicroarrays.The candidatemiRNAsaregenerallysubjectedtofurthervalidationby quantitativereversetranscriptionpolymerasechainreaction (qRT-PCR)in largercohorts. Toquantify it reliablyit is necessary to identifyendogenouscontrols.RNU-6B,RNU-48,and miR-16are commonlyusedfor thisaim,butnodefinitivecontrolgene has beenestablished[193,194].Recentstudieshavesuggestedmore reliableendogenouscontrolsforthequantificationofcirculating miRNAs.Chenetal.reportedthatacombinationoflet-7d,let-7g andlet-7iservesasanendogenouscontrolofserummiRNAsand it issuperiortothecommonlyusedreferencegenes[195].Kok etal.suggestednormalizationpanelsforthebetterquantification ofcirculatingmicroRNAsbyRT-qPCR[196].
Currentlythereareseveralstudiesfocusingontheuseof miR-NAsasdiagnosticandprognosticbiomarkersfordifferenttypes oftumors,suchascolon,prostate,breast,lung,andhematologic cancers.Intheliteratureithasbeenreportedthatanaberrant alter-nationofmiRNA wasobserved intheserumofNSCLCpatients, ascomparedtohealthycontrolsbyusingSolexiadeep
sequenc-ing[197];theauthorsaffirmthatmiR-25andmiR-223couldbe
usedasmarkersfordiagnosingNSCLC.Thecurrentlyknown circu-latingmiRNAsignaturesforNSCLCcancerincludeseveralmiRNA panelsandarrays[198].Recentresearchhasalsoshownthat cir-culating miRNAscan play an importantrole alsoas prognostic markers for NSCLC patients. For instance, a four miRNA panel (miR-486,miR-30d,miR-1andmiR-499)wasrelatedtotheoverall survival ofNSCLCpatientsundergoingsurgery,aswellas adju-vantchemotherapy[199],andincreasedserumlevelsofmiR-125b inNSCLCpatientswereassociatedwithnon-responsive cisplatin-basedchemotherapy[200].
SomestudieshaveevaluatedtheuseofcirculatingmiRNAsas biomarkersformonitoringtheresponsetotherapyalsoforbreast cancercells;infact,manystudiesconfirmedthatcirculating miR-155waselevatedintheserumofbreastcancerpatients[201].The alteredlevelsofcirculatingmiRNAscanalsobeusedtopredictthe effectofchemotherapy;forexample,elevatedlevelsofmiR-125bin breastcancerpatientscanindicatealowertherapeuticresponseto 5-Florouracil(5-FU),epirubucinorcyclophosphamide(FEC)[202]. StudieshavealsoconsideredtoemploycirculatingmiRNAsas diagnosticandprognosticmarkersforCRC.miR-92wasfoundtobe significantlyelevatedintheplasmaofCRCpatientsandintumor tissues,ascomparedtotheirnormalcounterparts,suggestingthat thismiRNAcouldbeusedasapotentialnon-invasivemarkerfor CRCdiagnosis[203].SeveralcirculatingmiRNAshavebeenshown toberelatedtoCRCmetastasis.AhighlevelofserummiR-200c showedasignificantcorrelationwiththelymphnodeanddistant metastasis,therebysuggestingthatitcouldbeusedasanindicator forpredictingCRCmetastasis[204].Overall,thesestudiesallowto affirmthatcirculatingmiRNAscouldserveaspromisingmarkers forcancerdiagnosisandprognosis.
7. Challenges,opportunitiesandpitfallsofmiRNAprofiling
Tissuebiopsiesarestillconsideredthegoldstandardfor molec-ularevaluationofcancer,althoughthisprocedureisinvasiveand hasseverallimitsrelatedtothelowpossibilityofsamplingandto theheterogeneityoftissues,thereasonwhybiopsyonlygivesa
smallsamplingoftheentiretumor.Theselimitationsaccountfor thepotentiallymissingclinicalinformation comingfrombioptic tissues.Moreover,anothersevererestrictionisrepresentedbythe littleopportunitytogainmultiplesamplesfollowingthevarious treatmentcycles,thusprecludingthepossibilitytoanalyzetumor evolutionovertimeandinresponsetotherapy.Inaddition,for cer-tainmalignanciesitisimpossibletoaccesstothetissueduetothe localization.Thisseriesofissueshasgeneratedtheboosttofind alternativemethodsforearlydetection,diagnosisandtherapeutic monitoringofcancer.
miRNAprofilingcanprovidearichamountofbiological infor-mation,inviewofthemultiplicityofmessangerRNAsthatmay bemodulatedasaconsequenceoftheprecisealterationinmiRNA expressionpattern.Themostimportantapplicationsrangefrom theanalysisofawidevarietyofphysiologicalprocesses–suchas organismsdevelopment,aswellasestablishmentandpreservation oftissuedifferentiation–tothemonitoringofpathological condi-tions–ascancer,cardiovascularandautoimmunediseases–for whichmiRNAsarebecominghelpfulbiomarkersandinnovative reagentsforre-programmingcellfateintherapeuticapplications
[205].AconsiderableinterestinmiRNAprofilinghasbeen
devel-opedalsointheforensicfield,wherehighlysensitivemeasurement ofthesemoleculesmayprovideinformationsuchasthecellular compositionofinquiredsamples.Abigsourceofinformationabout generegulationmayderivefromthecombinedanalysisofmiRNAs, mRNAsandproteinprofileonagenomicscale,althoughmiRNAs aremuchmorestablethanmRNAsinabroadvarietyofspecimens –includingcelllines,blood,plasma,serum,urine,freshtissuesand formalin-fixedtissueblocks–andarealsoquantifiablewithhigher sensitivitythanproteins, sincetheycanbeamplifiedbyRT-PCR
[206].SampleprocessingandRNAextractionmethodsplaya
lead-ingroleinthesuccessfuloutcomeofmiRNAprofiling,especiallyfor samplessusceptibletodegradation[207,208].Aspreviouslysaid, miRNAscanbeextractedwithhighqualityfromseveralcelland tissuesources[209];theisolationmethodsaregenerallythesame asfortheisolation oftotalRNA,apartfromthat protocolsused formiRNAisolationcanbemodified,tosomeextent,inorderto retainand/ortoenrichthesmallRNAfraction[209];theglobalyield inmiRNAfromfreshtissuesandcelllinesisgenerallygoodand suitableforprofilingstudies.Instead,concerningmiRNAcoming fromformalin-fixedparaffinembeddedtissues(FFPE),itis surpris-inglystableandintactindependentlyfromformalinfixationtime anddurationoftissueblockstorage[210],differentlyfrommRNA, whichisgenerallyfragmentedandlessreliableinFFPEcomparedto freshtissue.ThisstabilityconferstomiRNAsagreatadvantageover mRNAsastissuemarkersintheclinicalsetting,whereFFPEis gen-erallytheonlykindofsampleavailable.Someissuesmayarisein thecaseofmiRNAextractionfromsamplesthatrequirethetuning ofprotocolsaimedatoptimizingtheefficiencyandatincreasingthe qualityofproducts.Achallengingspecimenisrepresentedbybody fluids,wheretherearehighlevelsofendogenousRNasethatmay rapidlyinactivatemiRNAsinthecaseofextractioncarriedoutwith methodsthatfailtocompletelyinactivateRNase[211].Moreover, thequalityandtheamountofmiRNAextractedfrombloodmayalso beinfluencedbyothervariablesascentrifugationsettings,white bloodcellcounts,andredbloodcellhemolysis[212].Therefore,it isstrictlynecessary,forreproducibilityandaccuratenessofresults, toassessbothintegrityand yieldofextractedmiRNAbymeans ofspectrophotometry and/orautomated capillary electrophore-sisinstruments;anotherstrategyaimedatverifyRNAextraction efficiency,consistsintheaddition,intheearlystageofmiRNA iso-lation,ofaknownamountofsyntheticmiRNAsunexpressedin thebiologicalsample[213],sincetheirmeasurementcangive use-fulinformationabouttheaccuracyoftheextractionmethodand thepreservationofmiRNAintegrity.Detectionandquantification undergoalsootherissuescorrelatedwiththeshortnessofmature
miRNAandtheconsequentdifficultyencounteredinthe anneal-ingtotraditionalprimersdesignedforreversetranscriptionand
PCR[214];moreover,theabsenceofacommonsequence,suchas
poli(A)-tailformRNA,furthercomplicatesthepossibilityofa selec-tiveenrichment,thatisnecessary,giventhescarceabundanceof miRNAfraction(∼0.01%)respecttothetotalamountofRNA[215]
andtheexistenceofmiRNAfamiliesandvariantswhosemembers differeachotherfor singleor fewnucleotides.Inspiteofthese issues,theexigencyofprofilinghasgivenrisetothedevelopment ofthreemainapproaches–qRT-PCR,hybridisation-based meth-ods(e.g.,DNAmicroarrays)andhigh-throughputsequencing(i.e., RNAseq)–whichallowthedefinitionoftissue-basedand circulat-ingmiRNAbiomarkerswithhighaccuracy[216,217].
8. Conclusions
Theremarkableimportanceofanearlycancerdiagnosis, moni-toringandtreatment,inviewofanefficientpatient’smanagement, hasprovidedastrongboosttotherecognitionoftumor-specific, noninvasive,and easytodetectand quantifybiomarkers.Both blood-basedandtissuebiopsies-obtained proteinmarkers,until nowconsideredthegoldstandardformolecularevaluationof can-cer,haveshownlimitedspecificityandsensitivity.Inthisreview wehave focusedonthepossibility tocomplement theexisting biomarkerswiththedetectionofspecificncRNAsfromcancer tis-suesand,moreimportantly,wehaveanalysedtheopportunityto obtainapanelofclinicalrelevantcirculatingncRNAsfora mini-mallyinvasivediagnosisandmonitoringofneoplasms.Basedonthe functionalroleofnonprotein-codingportionofgenomeinalarge numberofmechanismsofgeneregulation–fromthetransmission oftransgenerational epigeneticinformation totheregulationof normalphysiologyandhumandiseases–wehavehereinreported avarietyofmiRNAsandlncRNAsparticularlyderegulatedin pre-clinicalandclinicalcancermodels,analysingtheirinhibitoryor promotingroleintheregulationofsolidandhematologic malig-nancies. We have also described the most common methods employedforncRNAdetectionandquantificationinbodyfluids focusingontheiradvantagesandlimitations;theexpressionprofile ofcirculatingmiRNAsis,infact,highlyinfluencedbysample prepa-rationmethods.IssuesarisingfromncRNAextractionrequirethe fine-tuningofprotocolsfortheoptimizationoftheefficiencyand fortheimprovementofbothamountandqualityofproducts. Any-way,apromisingperspectivecomesfromtheemployofexosomal miRNAsforvariousclinicalapplications,includingtreatmentand diagnosisofneoplasms;exosomalcargomay,infact,specifically reflectthepathogenesisofthedonorcellthatcanbetransferred torecipientcellsalteringtheirmolecularcharacteristicsanditcan alsobeeasilydetectedinbodyfluidsprovidingprecious informa-tionwithanon-invasiveapproach.Moreover,anothertopichere coveredistherecognitionofncRNAsaspromisingtherapeutic tar-getsforanticancertreatmentsandtheneedtoincreasetheirtime ofcirculationintheblood,aswellastopotentiatetheir preferen-tialaccumulationincancertissues.Therapeuticstrategiesaimedat modulatemiRNAsexpressionarerecentlyemergingjustinvirtue ofncRNAs’abilitytoinfluencecellularbehaviour[218].miRNAs cancontributetotumorinitiationandprogressionthrough sev-eralmechanismsand,therefore,multipletherapeuticapproaches havebeenproposedtotargettheseprocesses.Indetail,the inhi-bitionofoncogenicmiRNAscanbeobtainedbyusingantisense oligonucleotides(ASOs)thatareabletoadheretothemiRNAtarget formingmiRNA-anti-miRNAbindingcomplexes.Thesemolecules canbegroupedintothreemajorcategories:antagomirs,locked nucleicacids(LNAs)andASOswithchemicalalterationsto opti-mizeefficacy.Anothertherapeuticapproachforreducingthelevels ofoncogenicmiRNAsconsistsintheuseofcompetitiveinhibitors,
suchas vectors bearing multiple artificialmiRNA binding sites underthecontrolofstrongpromoters.Asregardsoncosuppressor miRNAs,restorationoftheirbasallevelsisthemostusedstrategy, whichcanbeobtainedeitherbyusingmiRNAmimics,oravailingof theso-calledepigeneticmodifiers-thatactonhypermethylationand histonemodificationpatterns,whichhavebeenlinkedtomiRNA dysregulation-andenhancersofthemiRNAprocessingmachinery - anotherclass of smallmolecules that has beendeveloped to enhanceRNAiandtoinducemiRNAprocessing[218,219].These requirementshavestimulatedthedevelopmentofbothviraland non-viraldeliverysystemsandsomeofthemhavedemonstrated promisingresultsonhumans.Certainly,deliverystrategiesforthe employofncRNA-baseddrugsinclinicalpracticeandncRNA pro-filingfortheidentificationofnovelbiomarkers,stillrepresentan importantchallengeforbothcliniciansandresearchersandrequire furtherimprovementsforasuccessfulclinicaltranslation. How-ever,theencouragingpremisesderivedfromthegrowinginterest provenbyscientificcommunityofferanexcellentperspectivefor thenearfuture.
Conflictofinterest
None.
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