1、OpinionTacklingAgingbyUsingmiRNAasaTargetanda ToolWing-Fu Lai,1,2,*Marie Lin,2and Wing-Tak Wong1miRNA is a class of short noncoding RNA that regulates gene expression atthe post-transcriptional level.Evidence of age-associated changes in miRNAexpression has been collected in models ranging from nema
2、todes to humans;however,there has been little discussion of how to turn our knowledge ofmiRNA biology into antiaging therapy.This opinion article provides a snapshotof our current understanding of the roles of miRNA in modulating the agingprocess.We discuss major chemical techniques for modifying th
3、e miRNAstructure as well as developing delivery systems for intervention.Finally,technical needs to be met for bench-to-clinic translation of miRNA-basedinterventions are highlighted for future research.Genetic Manipulation to Combat AgingThe process of aging is caused not only by the accumulation o
4、f mutations that hamper properfunctioning of normal genes but also by age-associated alterations at the epigenetic level.Thelatter results from a variety of changes ranging from DNA methylation to chromatin remodeling.For many years,diverse approaches have been reported for genetic manipulation,incl
5、udingthe use of small interfering RNA(siRNA)for gene silencing and expression vectors for geneupregulation.Among these,microRNA(miRNA),which is short and noncoding single-strandedRNA of 1825 nt in length,displays promising application potential because it not only can beused to mediate gene silencin
6、g but also plays regulatory roles in different biological processes(e.g.,cell proliferation,apoptosis,development,and differentiation)18.Alterations in miRNAexpression are related not only to age-related diseases 9,10 but also to aging per se 1114.This was demonstrated by an earlier study on periphe
7、ral blood mononuclear cells(PBMCs,see Glossary)where a cohort of 21 different miRNA molecules were found to beupregulated during human aging,and 144 miRNA molecules were suppressed 15.Thisstudy suggested the feasibility of using miRNA as an endogenous therapeutic target for thedevelopment of antiagi
8、ng interventions.miRNAidentificationhastraditionallyemployedhybridization-basedarraymethods.Theefficiencyisfar from satisfactory,and this has impeded the development of miRNA-based antiaging therapy.Recently,the increased accessibility and affordability of commercial platforms(e.g.,the ABISOLiDsyste
9、m,theRoche454/FLXsystem,andtheIlluminaGenomeAnalyzer)forparallelsequenc-inghasfacilitatedhigh-throughputscanningofchangesinmiRNAexpression16,17.Theefficiencyof identifying the targets of miRNA has been further facilitated by using HITS-CLIP(i.e.,high-throughput sequencing of RNA isolated by crosslin
10、king immunoprecipitation)1820 andPAR-CLIP(i.e.,photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation)21,22.Nevertheless,few studies have addressed the feasibility and technical demands of turningour expanding knowledge of miRNA biology into antiaging therapy.Aging is a comple
11、x processtaking place at cellular,tissue,and organ levels;however,in this article we restrict our discussionsto aging at the cellular level.Recognizing that cells are the most fundamental units of an organism,HighlightsThe accumulated knowledge of miRNAbiology has established a foundationfor using m
12、iRNA as a target and atool for the development of antiaginginterventions.The identification of miRNA for antiaginginterventions has been facilitated bythe use of in silico methods to identifyhairpin structures in noncoding andnonrepetitiveregionsingenomesequences.DiversemiRNAanalogsincludingphosphor
13、othioateoligodeoxynucleotides(ODNs),locked nucleic acid(LNA)oligo-nucleotides,and peptide nucleic acids(PNAs)havebeengeneratedtoenhancethe gene-silencing efficiency of miRNAtherapeutics.Both viral and nonviral systems havebeen developed to deliver miRNA thera-peutics,and some allow real-timetracking
14、 of the delivery process andcodelivery of multiple agents.1Department of Applied Biology andChemical Technology,Hong KongPolytechnic University,Hong KongSpecial Administrative Region,China2Health Science Centre,ShenzhenUniversity,Shenzhen,China*Correspondence:rori0610graduate.hku.hk(W.-F.Lai).Trends
15、 in Molecular Medicine,August 2019,Vol.25,No.8https:/doi.org/10.1016/j.molmed.2019.04.007673 2019 Published by Elsevier Ltd.Trends in Molecular Medicinewe discuss the major principles for designing miRNA structures,followed by a discussion onthe possible use of miRNA as a target and a tool in modula
16、ting the aging process in cells,whichrepresents a new direction in the future development of antiaging therapeutics.Strategies to Enhance miRNA PerformancemiRNA-mediated gene silencing generally involves specific binding of miRNA to the miRNA-induced silencing complex(miRISC)(Box 1).To enhance this
17、binding,various strategieshave been proposed to chemically modify miRNA molecules.Owing to the presence ofRNases,naked miRNA in the bloodstream is degraded very easily 23.To improve its stability,several chemical derivatives of RNA molecules have been developed.Phosphorothioateoligodeoxynucleotides(
18、ODNs),in which one of the nonbridging oxygens in the phosphategroup is replaced with sulfur 24,are a good example.Compared with naked miRNA,thesedisplay increased resistance to nuclease degradation 25,26.Unfortunately,their half-lives aregenerally still very short.For example,the half-lives of penta
19、decamers in adult human serumandinthecellularpost-mitochondrialextractarelessthan10h24.Inaddition,giventheirnonspe-cific effects on cell growth inhibition and their low affinity for mRNA,phosphorothioate ODNs arenot optimized for therapeutic use 27.To reduce clearance from tissues,2-O-methoxyethylmo
20、dificationhasbeenincorporatedintothebackboneofthephosphorothioateODN25.Morere-cently,2-O-methyl groups have been introduced into the ribose moiety of the phosphorothioateODN 28.The 20-mer ODN generated has reduced nonspecific inhibitory effects on cell growth,and has effectively targeted sites 109 a
21、nd 277 of Bcl2 mRNA 28.These modifications greatlyenhance the potential of phosphorothioate ODNs in therapeutic applications.In addition to phosphorothioate ODNs,other examples of RNA derivatives include lockednucleicacid(LNA)oligonucleotides29 and peptide nucleic acids(PNAs)30.The clinicalapplicabi
22、lity of LNA oligonucleotides has been demonstrated by Miravirsen(Santaris Pharma,Hrsholm,Denmark),and clinical trials have been undertaken to evaluate their possible use intreating hepatitis C virus(HCV)infection(ClinicalTrials.gov identifier NCT01200420)31,32.PNAs are uncharged oligonucleotide anal
23、ogs in which the sugarphosphodiester backbonehas been substituted by an achiral structure that consists of N-(2-aminoethyl)-glycine units.Byusing standard bioconjugation techniques,these derivatives can be incorporated with variousfunctional moieties(including targeting ligands,proteins,and peptides
24、)to enhance tissuespecificity and prolong half-lives 3335.Further optimization will be necessary to realize theirpotential as future therapeutics.Working Principles of InterventionTodevelopantiagingtherapybasedonmiRNA,twodirectionsareavailable(Figure1,KeyFigure).The first is to use miRNA to silence
25、the expression of genes whose silencing can lead to agingretardation or even lifespan prolongation.The second is to exploit miRNA as an endogenoustherapeutic target.Here age-related changes in the expression of endogenous miRNAare first identified,followed by intervention to combat those changes.To
26、counteract the decliningexpression level of an endogenous miRNA molecule,one strategy is to administer anoligonucleotide mimic(that has the same sequence as the mature endogenous miRNA andcan bind to the RISC).Although single-stranded RNA molecules can be used as mimics,double-stranded mimics,which
27、have a guide strand and a passenger strand,generally havehigher potency 36.If the expression of an endogenous miRNA molecule increases withadvanced age,the use of an anti-miRNA oligonucleotide(AMO)complementary to the miRNAmature strand can prevent the miRNA molecule from inactivating the target mRN
28、A transcript.Since the turn of the past century,miRNA sponges have emerged as a new tool to modulatethe activity of endogenous miRNA.Not only can these sponges serve as decoys to modulatethe activity of overexpressed miRNA molecules,but they may also be used in long-term loss-GlossaryGene therapy:a
29、procedure tomanipulate the genetic component of aliving cell to tackle or improve thedisease condition of a patient.Hayflicklimit:the maximumnumber oftimes a cell can divide.Immunoprecipitation:a technique toprecipitate a protein antigen from asolution by using an antibody that bindsto that protein
30、antigen.Liposomes:spherical vesicles thatpossess one or more lipid bilayers.Theyhave been exploited extensively for usein therapeutics delivery.Locked nucleic acid(LNA)oligonucleotides:RNA analogs inwhichtheribosemoietyislockedusingabridge that connects the 4-carbon and2-oxygen atoms in an RNA-mimic
31、kingN-type(C3-endo)conformation.miRNA sponges:RNA molecules thatpossess repeated miRNA antisensesequences that can sequester miRNAmolecules away from their endogenoustargets.Peptide nucleic acid(PNA):a DNAmimic that possesses a pseudopeptidebackbone.Peripheral blood mononuclear cells(PBMCs):peripher
32、al blood cells thathave a round nucleus.Examples includelymphocytes and monocytes.Quantum dots(QDs):highlyfluorescent semiconductor nanocrystalsthat exhibit a quantized energyspectrum.Telomere:a segment of DNA at theend of a chromosome that givesprotection to that chromosome.Trends in Molecular Medi
33、cine674Trends in Molecular Medicine,August 2019,Vol.25,No.8of-function studies 37.Although more research will be necessary to determine whether in vivosponge expression is a valid alternative to genetic knockouts of miRNA families,miRNA spongeshave the potential to manipulate multiple miRNA molecule
34、s simultaneously.To develop a feasible miRNA-based intervention,boosting the potency of the miRNA moleculeper se is required,but the availability of effective carriers for delivering the miRNA molecule intotarget sites is equally important.The most extensively used carriers are viral vectors 38.Viru
35、sesadopted for gene delivery include retroviruses,adenoviruses,and adeno-associated virusesBox 1.miRNA Biogenesis and Mechanism of ActionmiRNA modulates gene expression at the post-transcriptional level via binding complementarily to the coding region or 3 untranslated region(UTR)of the target mRNAt
36、ranscript.Biogenesis of miRNA starts with the generation of pri-miRNA,the primary miRNA precursor.The pri-miRNA transcript is a capped,polyadenylated RNAstrand.A double-stranded stem-loop structure is subsequently formed(Figure I).Under the action of DGCR8 and Drosha in the nucleus,pri-miRNA is proc
37、essed intopre-miRNA,a hairpin structure of 70100 nt.With the help of exportin-5(a RanGTP-dependent double-stranded RNA-binding protein),pre-miRNA is exported to thecytosol,where it is further processed into a double-stranded miRNA duplex under the action of Dicer 121,122.Binding to the miRISC is an
38、important step that deter-mines the effective action of miRNA molecules 123.After the duplex binds to the miRISC,it unwinds into two strands:the passenger strand and the mature strand124.The former is released and degraded,whereas the latter remains bound to the miRISC to silence the expression of a
39、 target gene by inhibiting mRNA translationor by inducing degradation of the mRNA transcript.Because perfect pairing is not essential for the miRISC to act on the target mRNA transcript,one miRNA moleculemay act on several mRNA targets.Such promiscuity enables one miRNA molecule to regulate the expr
40、ession of multiple genes.TrendsTrends inin MolecularMolecular MedicineMedicineFigure I.miRNA Biogenesis and Mechanism of Action.Abbreviations:Pre-miRNA,precursor miRNA;Pri-miRNA,primary miRNA;RISC,RNA-inducedsilencing complex.Trends in Molecular MedicineTrends in Molecular Medicine,August 2019,Vol.2
41、5,No.8675Key FiguremiRNA as a Target and a Tool.TrendsTrends inin MolecularMolecular MedicineMedicineFigure 1.Todevelop anmiRNA-based therapy,thetherapeuticmiRNA isfirstselected.Alternatively,age-relatedchanges inendogenousmiRNA expressionareidentified.Adeliverysystem isthen employed todeliver thera
42、peuticswhich either counteractchanges in miRNAexpression or degrade the mRNAtranscript ofa targetgene in aged cells.Dotted arrows indicate the action of the therapy;solid arrows indicate the progression of stages and biological levels.Abbreviation:Pre-miRNA,precursor miRNA.Trends in Molecular Medici
43、ne676Trends in Molecular Medicine,August 2019,Vol.25,No.8(AAVs).The clinical potential of viralvectors,however,is constrained by the safetyrisksof viruses.Thesehavebeendocumentedin reportsonthedevelopmentofleukemiainX-linked severe com-bined immunodeficiency(X-SCID)patients treated with gammaretrovi
44、ral vectors 38,and thedeath of an 18-year-old patient who was administered an adenoviral vector for the treatment ofinherited enzyme deficiency 39.Other instances of preneoplastic or truly neoplastic cell expan-sion caused by insertional mutagenesis have also been observed in gene therapy of Wiskott
45、Aldrich syndrome(WAS)40 and X-linked chronic granulomatous disease 41.This emphasizesthe need to develop nonviral alternatives for delivering nucleic acid therapeutics.Over recent decades different nonviral delivery methods ranging from lipofection to electropora-tion have been developed(Table 1)425
46、0.In addition to their higher tolerance to cargo size,these methods are generally less immunogenic and pathogenic.Till now liposomal systemsTable 1.Examples of Technologies That May Be Applicable to Delivery of miRNA TherapeuticsApproachSystemWorking principleExampleRefsBiologicalmethodViral vectorA
47、 viral vector,in which the capacity of viral replication ismaintained but most genes coding for viral proteins areremoved,is employed for carrying miRNA therapeuticsA lentiviral vector expressing miR-15a/16 increased thelevel of miR-16 in serum after systemic administration toa de novo New Zealand b
48、lack(NZB)mouse model.ThemiR-16 relative quantification(RQ)value of the plasmafrom treated mice was increased by 50%relative to miceinjected with the control GFP-expressing lentiviral vector42Delivery of miR-26a to hepatocellular carcinoma(HCC)cells using AAVs reduced the level of miR-26aexpression,r
49、esulting in inhibition of tumor growth andinduction of apoptosis43ExosomeVirus-infected cells are used to generate exosomes forcarrying miRNA therapeuticsDelivery of an miRNA-155 inhibitor to RAW macrophagesusing exosomes reduced lipopolysaccharide(LPS)-induced tumor necrosis factor(TNF)-productiona
50、nd partially inhibited the LPS-induced decrease in theSOCS1 mRNA level44ChemicalmethodCationicpolymerCationic polymers are used to complex with negativelycharged miRNA molecules to form polyplexes for RNAdeliveryPolyplexes formed between chloroquine-containing2-(dimethylamino)ethyl methacrylate copo