非編碼RNA與腫瘤發生發展《癌生物學》非編碼RNA與腫瘤發生發展《癌生物學》Anartist'sconceptionshowsanRNAmolecule,whichmayhaveservedasanearlyformoflifeonEarth.NicolleRagerFuller2Anartist'sconceptionshowsa

LUCA：LastUniversalCommonAncestor

LUCA：LastUniversalCommonA4TheRNAWorld,4theditionColdSpringHarborLaboratory(CSHL)Press2006201019991993RaymondF.GestelandThomasR.CechJohnF.AtkinsEditedBy4TheRNAWorld,4theditionCol5RegulatoryRNAs↗

SmallRNAmoleculesregulategeneexpressionbypost-transcriptionalgenesilencing.↗

Xist:thefirstlongnon-codingRNAregulatinggeneexpression.↗

Non-codingRNAscontrolepigeneticphenomena.…http://www.mfpl.ac.at/rna-biology/rna-history/5RegulatoryRNAs↗

SmallRNAmoNon-codingRNAsinHumanGenome6?dkfz.de組成型非編碼RNA

rRNA，tRNA，snRNA，snoRNA調控型非編碼RNA

微小非編碼RNA

microRNA,

siRNA,piRNA,etc長非編碼RNALongnon-codingRNAs(LncRNAs)人類基因組（30億堿基對）

>70%被轉錄

<2%轉錄后表達蛋白(mRNA)環狀RNACircularRNA(circRNAs)人類基因組蘊藏著數目巨大的非編碼RNANon-codingRNAsinHumanGenomNon-codingRNAsandCancer7DouglasHanahanandRobertA.Weinberg,Cell144,2011:646-674.OncogenicandTumorsuppressivencRNAsNon-codingRNAsandCancer7DouContents8SmallRNAs(microRNAs);

Longnon-codingRNAs(lncRNAs);

CircularRNAs(circRNAs).Contents8SmallRNAs(microRNAMicroRNAdiscovery1993年，第一個miRNA基因——lin-4(秀麗線蟲)(Lee,etal.Cell,1993,75:843-854)2000年，第二個miRNA基因——let-7(秀麗線蟲)(Reinhart,etal.Nature,2000,403:901-6)2001年，德、美等三個實驗室發現約100個miRNA基因(果蠅、線蟲以及人類等)(Lagos-Quintana,etal;Lau,etal;Lee,etal.Science)/index.shtml

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

35828maturemiRNAsin223speciesHomosapiens：1,881precursor(2,588)MicroRNAdiscovery1993年，第一個miRDNACapAAAAAAAAAApri-miRNAmicroRNAExonIntronDNACapAAAAAAAAAAATGTGAmRNAProtein轉錄轉錄翻譯加工microRNA:由19-25個核苷酸組成的非編碼小分子RNA10DNACapAAAAAAAAAApri-miRNAmicroBiogenesisandActionofmicroRNAs11BiogenesisandActionofmicroMicroRNA-mRNAcomplexinterplayOnemiRNAtargetsmultiplemRNAsOnemRNAbymultiplemiRNAs12MicroRNAscanregulateabout~60%ofallmRNAs!MicroRNA-mRNAcomplexinterplaMicroRNARegulationofCancerBiology13miR-125b,HepatologymiR-210HepatologymiR-423CarcinogenesismiR-151NatureCellBiologymiR-195,HepatologymiR-194,ScienceSignalingmiR-30d,HepatologymiR-124GastroentolgoymiR-199HepatologyMicroRNARegulationofCancerMicroRNA領域值得關注的幾個問題141.

microRNA生物合成及表達調控機制；2.microRNA在疾病進程中的作用及調控網絡；（疾病模型）3.microRNA在生理、病理中的調節作用；（KO，TG）4.microRNA轉化醫學研究：血清診斷、預后判斷、分子分型、治療探索等MicroRNA領域值得關注的幾個問題141.microContents15

SmallRNAs(microRNAs);

Longnon-codingRNAs(lncRNAs);

CircularRNAs(circRNAs).Contents15SmallRNAs(microRNLncRNADiscovery16LncRNA是一類轉錄本長度超過200nt的RNA分子1980s-1990s:IndividuallncRNAs(eg,XistandH19)arediscoveredthroughtraditionalgenemappingapproaches;

Early2000s:LargescalecDNAsequencingleadstothediscoveryofasurprisingnumberoflncRNAtrancscripts;

2000s-Now:ThenumberofpredictedproteincodinggenesinhumangoesdownandthenumberofdetectedlncRNAtranscriptsincreases.GENECODEV21LncRNADiscovery16LncRNA是一類轉錄本LncRNA分類和特征17根據基因組位置分為5類：

雙重活性：可結合蛋白與核酸（RNA,DNA）TrendsGenet.2014Oct;30(10):439-52.LncRNA具復雜的二級與高級結構，決定其分子調控多樣性NatRevGenet.2014Jan;15(1):7-21.Sense,Antisense,Intronic,Intergenic,BidirectionalLncRNA分類和特征17根據基因組位置分為5類：雙重LncRNA功能和作用機制18ModelsofnuclearlncRNAfunctionModelsofcytoplasmiclncRNAfunctionNatRevGenet.2014Jan;15(1):7-21.mRNA穩定性mRNA翻譯miRNAsponge蛋白穩定性/修飾染色質重塑表觀遺傳修飾基因轉錄可變剪接LncRNA具有重要分子調節功能，而不是“轉錄的噪音”LncRNA功能和作用機制18Modelsofnucle19BiochimBiophysActa.2014;1839(11):1097-109.LncRNA與癌癥密切相關OncogenicandTumorsuppressivelncRNAs19BiochimBiophysActa.2014;12020HOXlincRNAsaresystematicallydysregulatedinbreastcancerHOXlincRNAsaresystematicalThelincRNAHOTAIRisincreasedinexpressioninprimarybreasttumoursandmetastasesThelincRNAHOTAIRisincrease

HOTAIRhasprognosticvalueformetastasisandsurvivalHOTAIRhasprognosticvaluefHOTAIRpromotesinvasionofbreastcarcinomacells.HOTAIRpromotesinvasionofbrHOTAIRpromotesmetastasisofbreastcarcinomacells.HOTAIRpromotesmetastasisofThemechanismofHOTAIRinbreastcancerprogressionThemechanismofHOTAIRinbreLncRNA參考文獻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關鍵詞：腫瘤類型+lncRNALncRNA參考文獻27Longnon-codingRNContents28SmallRNAs(microRNAs);Longnon-codingRNAs(lncRNAs);

CircularRNAs(circRNAs).Contents28SmallRNAs(microRN29CircularRNAWiluszandSharp,Science,2013ExonicCircularRNA(circRNAs,環狀RNA)CircularRNA(circRNA)isatypeofRNAthat,unlikelinearRNA,formsacovalentlyclosedcontinuousloop.~100,000circRNAsinhuman29CircularRNAWiluszandSharpHistoryofcircularRNAH.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....發現（1976）拓展（1990s）

熱點（2010s）30HistoryofcircularRNAH.L.Sa31JuliaSalzmanSalzmanet.al.,PLoSone,201231JuliaSalzmanSalzmanet.al.32Memczaketal.,Nature,2013Hansenetal.,Nature,2013NikolausRajewskyMCD,Berlin,GermanyJ?rgenKjemsAarhusUniversity,Denmark32Memczaketal.,Nature,201333BiogenesisofcircularRNADirectbacksplicingExonskippingJeckandSharpless,NatureBiotechnology,201433BiogenesisofcircularRNADi34Liangetal.,GenesandDevelopment,2014Starkeetal.,CellReports,2014Ashwal-Flussetal.,MolecularCell,2014Ivanovetal.,CellReports,2015Connetal.,Cell,2015BiogenesisofcircularRNAZhangetal.,Cell,201434Liangetal.,GenesandDeve環狀RNA研究現狀及問題一、鑒定（高通量測序／生物信息分析）二、生物合成和調控三、功能和機制四、意義RNA前體剪切加工的副產物？對細胞功能？機制？相對穩定，是否作為疾病診療靶點？如何生成？!

出核？去向（代謝）？鑒定方法；組織、癌癥相關環狀RNA的存在情況？數量？表達？35環狀RNA研究現狀及問題RNA前體剪切加工的副產物？對細胞功RNA高通量測序鑒定環狀RNA36RNA高通量測序鑒定環狀RNA36人類正常和腫瘤組織中存在大量的環狀RNA人類正常和腫瘤組織中存在大量的環狀RNA38環狀RNA具有組織特異性，在腦組織高表達38環狀RNA具有組織特異性，在腦組織高表達NTNTallcancertissuesHCCtissues環狀RNA在腫瘤組織中存在差異表達NTNTallcancertissuesHCCtiss環狀RNA在腫瘤組織中具有重要的臨床意義環狀RNA在腫瘤組織中具有重要的臨床意義環狀RNA具有重要的功能環狀RNA具有重要的功能環狀RNA作用機制？環狀RNA作用機制？環狀RNA作用機制環狀RNA作用機制44SummaryandprospectmiRNA1990-00s～2000明確單一LncRNA1980-90s～30000待探索多樣復雜circRNA1970-90s～100000待探索未知

發現

數量

功能

機制44SummaryandprospectmiRNA19945SummaryandprospectmiRNAcircRNALncRNA45SummaryandprospectmiRNAcir46Questions？

CircularRNAisenrichedandstableinexosomes:apromisingbiomarkerforcancerdiagnosisQuestionsorContinued？CellRes.2015Aug;25(8):981-4.46Questions？QuestionsorCo47ThemetabolismofcircularRNAs?HighlystablePredominantlycytoplasmicExonucleases?47ThemetabolismofcircularR48Exosomes:cell-cellcommunicationsProteinmRNAmicroRNA?Note：exosomes≠nuclearexosome(RNasecomplex)Raposoet.al.,JCB,201330-100nm48Exosomes:cell-cellcommunic49cellsFBS-free,overnightUltracentrifugation300g×10min;15000rpm×20min;40000rpm×70minExosomesRNA-seqanalysisofcellandcell-derivedexosomesRNAsTransmissionelectronmicrograph49cellsFBS-free,overnightExos50MHCC-LM3cellsTotalRNAExosomesRNARibo(-)StrandedRNAseqUnmappedReadsBack-SplicedReadsCandidateCircularRNAsTopHat2AnchoralignmentCircularRNAsareenrichedinexosomes50MHCC-LM3cellsTotalRNAExoso51CircularRNAsareenrichedinexosomes51CircularRNAsareenrichedi52CircularRNAsareenrichedinexosomesCircularexonLinear-SplicedReads(L1)Linear-SplicedReads(L2)Back-SplicedReads(B)Back-SplicedRatio=B/(0.5XL1+0.5XL2)52CircularRNAsareenrichedi53CircularRNAssortingtoexosomesareregulatedCellularcircRNAsExosomalcircRNAsRegulated？53CircularRNAssortingtoexo54CircularRNAssortingtoexosomesareregulatedbyassociatedmiRNAs