非编码RNA与肿瘤发生发展课件

非编码RNA与肿瘤发生发展《癌生物学》Anartist'sconceptionshowsanRNAmolecule,whichmayhaveservedasanearlyformoflifeonEarth.NicolleRagerFuller2

LUCA：LastUniversalCommonAncestor4TheRNAWorld,4theditionColdSpringHarborLaboratory(CSHL)Press2006201019991993RaymondF.GestelandThomasR.CechJohnF.AtkinsEditedBy5RegulatoryRNAs↗

SmallRNAmoleculesregulategeneexpressionbypost-transcriptionalgenesilencing.↗

Xist:thefirstlongnon-codingRNAregulatinggeneexpression.↗

Non-codingRNAscontrolepigeneticphenomena.…http://www.mfpl.ac.at/rna-biology/rna-history/Non-codingRNAsinHumanGenome6©dkfz.de组成型非编码RNA

rRNA，tRNA，snRNA，snoRNA调控型非编码RNA

微小非编码RNA

microRNA,

siRNA,piRNA,etc长非编码RNALongnon-codingRNAs(LncRNAs)人类基因组（30亿碱基对）

>70%被转录

<2%转录后表达蛋白(mRNA)环状RNACircularRNA(circRNAs)人类基因组蕴藏着数目巨大的非编码RNANon-codingRNAsandCancer7DouglasHanahanandRobertA.Weinberg,Cell144,2011:646-674.OncogenicandTumorsuppressivencRNAsContents8SmallRNAs(microRNAs);

Longnon-codingRNAs(lncRNAs);

CircularRNAs(circRNAs).MicroRNAdiscovery1993年，第一个miRNA基因——lin-4(秀丽线虫)(Lee,etal.Cell,1993,75:843-854)2000年，第二个miRNA基因——let-7(秀丽线虫)(Reinhart,etal.Nature,2000,403:901-6)2001年，德、美等三个实验室发现约100个miRNA基因(果蝇、线虫以及人类等)(Lagos-Quintana,etal;Lau,etal;Lee,etal.Science)/index.shtml

(miRBaseSangerCenter)9Release21:June,2014miRNAcount:28645entries

35828maturemiRNAsin223speciesHomosapiens：1,881precursor(2,588)DNACapAAAAAAAAAApri-miRNAmicroRNAExonIntronDNACapAAAAAAAAAAATGTGAmRNAProtein转录转录翻译加工microRNA:由19-25个核苷酸组成的非编码小分子RNA10BiogenesisandActionofmicroRNAs11MicroRNA-mRNAcomplexinterplayOnemiRNAtargetsmultiplemRNAsOnemRNAbymultiplemiRNAs12MicroRNAscanregulateabout~60%ofallmRNAs!MicroRNARegulationofCancerBiology13miR-125b,HepatologymiR-210HepatologymiR-423CarcinogenesismiR-151NatureCellBiologymiR-195,HepatologymiR-194,ScienceSignalingmiR-30d,HepatologymiR-124GastroentolgoymiR-199HepatologyMicroRNA领域值得关注的几个问题141.

microRNA生物合成及表达调控机制；2.microRNA在疾病进程中的作用及调控网络；（疾病模型）3.microRNA在生理、病理中的调节作用；（KO，TG）4.microRNA转化医学研究：血清诊断、预后判断、分子分型、治疗探索等Contents15

SmallRNAs(microRNAs);

Longnon-codingRNAs(lncRNAs);

CircularRNAs(circRNAs).LncRNADiscovery16LncRNA是一类转录本长度超过200nt的RNA分子1980s-1990s:IndividuallncRNAs(eg,XistandH19)arediscoveredthroughtraditionalgenemappingapproaches;

Early2000s:LargescalecDNAsequencingleadstothediscoveryofasurprisingnumberoflncRNAtrancscripts;

2000s-Now:ThenumberofpredictedproteincodinggenesinhumangoesdownandthenumberofdetectedlncRNAtranscriptsincreases.GENECODEV21LncRNA分类和特征17根据基因组位置分为5类：

双重活性：可结合蛋白与核酸（RNA,DNA）TrendsGenet.2014Oct;30(10):439-52.LncRNA具复杂的二级与高级结构，决定其分子调控多样性NatRevGenet.2014Jan;15(1):7-21.Sense,Antisense,Intronic,Intergenic,BidirectionalLncRNA功能和作用机制18ModelsofnuclearlncRNAfunctionModelsofcytoplasmiclncRNAfunctionNatRevGenet.2014Jan;15(1):7-21.mRNA稳定性mRNA翻译miRNAsponge蛋白稳定性/修饰染色质重塑表观遗传修饰基因转录可变剪接LncRNA具有重要分子调节功能，而不是“转录的噪音”19BiochimBiophysActa.2014;1839(11):1097-109.LncRNA与癌症密切相关OncogenicandTumorsuppressivelncRNAs20HOXlincRNAsaresystematicallydysregulatedinbreastcancerThelincRNAHOTAIRisincreasedinexpressioninprimarybreasttumoursandmetastases

HOTAIRhasprognosticvalueformetastasisandsurvivalHOTAIRpromotesinvasionofbreastcarcinomacells.HOTAIRpromotesmetastasisofbreastcarcinomacells.ThemechanismofHOTAIRinbreastcancerprogressionLncRNA参考文献27Longnon-codingRNAHOTAIRreprogramschromatinstatetopromotecancermetastasis.

Nature.2010Apr15;464(7291):1071-6.PMID:20393566；AfunctionalgenomicapproachidentifiesFAL1asanoncogeniclongnoncodingRNAthatassociateswithBMI1andrepressesp21expressionincancer.CancerCell.2014Sep8;26(3):344-57.PMID:25203321TheSTAT3-bindinglongnoncodingRNAlnc-DCcontrolshumandendriticcelldifferentiation.Science.2014Apr18;344(6181):310-3.PMID:24744378；Pubmed关键词：肿瘤类型+lncRNAContents28SmallRNAs(microRNAs);Longnon-codingRNAs(lncRNAs);

CircularRNAs(circRNAs).29CircularRNAWiluszandSharp,Science,2013ExonicCircularRNA(circRNAs,环状RNA)CircularRNA(circRNA)isatypeofRNAthat,unlikelinearRNA,formsacovalentlyclosedcontinuousloop.~100,000circRNAsinhumanHistoryofcircularRNAH.L.Sanger,G.Klotz,D.Riesner,H.J.Gross,A.K.Kleinschmidt.(1976)Viroidsaresingle-strandedcovalentlyclosedcircularRNAmoleculesexistingashighlybase-pairedrod-likestructures.Proc.Natl.Acad.Sci.USA,73pp.3852–3856.Coca-Prados,M-THaM.(1979)ElectronmicroscopicevidenceforthecircularformofRNAinthecytoplasmofeukaryoticcells.Nature

280:339–340.

...NigroJM,ChoKR,FearonER,KernSE,RuppertJM,etal.(1991)Scrambledexons.Cell

64:607–613.CapelB,SwainA,NicolisS,HackerA,WalterM,etal.(1993)Circulartranscriptsofthetestis-determininggeneSryinadultmousetestis.Cell

73:1019–1030....SalzmanJ,GawadC,WangPL,LacayoN,BrownPO.(2012)CircularRNAsarethepredominanttranscriptisoformfromhundredsofhumangenesindiversecelltypes.PLoSOne7(2):e30733.MemczakS,JensM,ElefsiniotiA,TortiF,KruegerJ,etal.(2013)CircularRNAsarealargeclassofanimalRNAswithregulatorypotency.Nature495(7441):333-8.HansenTB,JensenTI,ClausenBH,BramsenJB,FinsenB,etal.(2013)NaturalRNAcirclesfunctionasefficientmicroRNAsponges.Nature495(7441):384-8.ZhangXO,WangHB,ZhangY,LuX,ChenLL,YangL.(2014)Complementarysequence-mediatedexoncircularization.Cell.25;159(1):134-47....发现（1976）拓展（1990s）

热点（2010s）3031JuliaSalzmanSalzmanet.al.,PLoSone,201232Memczaketal.,Nature,2013Hansenetal.,Nature,2013NikolausRajewskyMCD,Berlin,GermanyJørgenKjemsAarhusUniversity,Denmark33BiogenesisofcircularRNADirectbacksplicingExonskippingJeckandSharpless,NatureBiotechnology,201434Liangetal.,GenesandDevelopment,2014Starkeetal.,CellReports,2014Ashwal-Flussetal.,MolecularCell,2014Ivanovetal.,CellReports,2015Connetal.,Cell,2015BiogenesisofcircularRNAZhangetal.,Cell,2014环状RNA研究现状及问题一、鉴定（高通量测序／生物信息分析）二、生物合成和调控三、功能和机制四、意义RNA前体剪切加工的副产物？对细胞功能？机制？相对稳定，是否作为疾病诊疗靶点？如何生成？!

出核？去向（代谢）？鉴定方法；组织、癌症相关环状RNA的存在情况？数量？表达？35RNA高通量测序鉴定环状RNA36人类正常和肿瘤组织中存在大量的环状RNA38环状RNA具有组织特异性，在脑组织高表达NTNTallcancertissuesHCCtissues环状RNA在肿瘤组织中存在差异表达环状RNA在肿瘤组织中具有重要的临床意义环状RNA具有重要的功能环状RNA作用机制？环状RNA作用机制44SummaryandprospectmiRNA1990-00s～2000明确单一LncRNA1980-90s～30000待探索多样复杂circRNA1970-90s～100000待探索未知

发现

数量

功能

机制45SummaryandprospectmiRNAcircRNALncRNA46Questions？

CircularRNAisenrichedandstableinexosomes:apromisingbiomarkerforcancerdiagnosisQuestionsorContinued？CellRes.2015Aug;25(8):981-4.47ThemetabolismofcircularRNAs?HighlystablePredominantlycytoplasmicExonucleases?48Exosomes:cell-cellcommunicationsProteinmRNAmicroRNA?Note：exosomes≠nuclearexosome(RNasecomplex)Raposoet.al.,JCB,201330-100nm49cellsFBS-free,overnightUltracentrifugation300g×10min;15000rpm×20min;40000rpm×70minExosomesRNA-seqanalysisofcellandcell-derivedexosomesRNAsTransmissionelectronmicrograph50MHCC-LM3cellsTotalRNAExosomesRNARibo(-)StrandedRNAseqUnmappedReadsBack-SplicedReadsCandidateCircularRNAsTopHat2AnchoralignmentCircularRNAsareenrichedinexosomes51CircularRNAsareenrichedinexosomes52CircularRNAsareenrichedinexosomesCircularexonLinear-SplicedReads(L1)Linear-SplicedReads(L2)Back-SplicedReads(B)Back-SplicedRatio=B/(0.5XL1+0.5XL2)53CircularRNAssortingtoexosomesareregulatedCellularcircRNAsExosomalcircRNAsRegulated？54