非編碼RNA與腫瘤發(fā)生發(fā)展課件

非編碼RNA與腫瘤發(fā)生發(fā)展《癌生物學(xué)》Anartist'sconceptionshowsanRNAmolecule,whichmayhaveservedasanearlyformoflifeonEarth.NicolleRagerFuller2

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

SmallRNAmoleculesregulategeneexpressionbypost-transcriptionalgenesilencing.↗

Xist:thefirstlongnon-codingRNAregulatinggeneexpression.↗

Non-codingRNAscontrolepigeneticphenomena.…h(huán)ttp://www.mfpl.ac.at/rna-biology/rna-history/Non-codingRNAsinHumanGenome6?dkfz.de組成型非編碼RNA

rRNA，tRNA，snRNA，snoRNA調(diào)控型非編碼RNA

微小非編碼RNA

microRNA,

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

>70%被轉(zhuǎn)錄

<2%轉(zhuǎn)錄后表達(dá)蛋白(mRNA)環(huán)狀RNACircularRNA(circRNAs)人類基因組蘊(yùn)藏著數(shù)目巨大的非編碼RNANon-codingRNAsandCancer7DouglasHanahanandRobertA.Weinberg,Cell144,2011:646-674.OncogenicandTumorsuppressivencRNAsContents8SmallRNAs(microRNAs);

Longnon-codingRNAs(lncRNAs);

CircularRNAs(circRNAs).MicroRNAdiscovery1993年，第一個(gè)miRNA基因——lin-4(秀麗線蟲)(Lee,etal.Cell,1993,75:843-854)2000年，第二個(gè)miRNA基因——let-7(秀麗線蟲)(Reinhart,etal.Nature,2000,403:901-6)2001年，德、美等三個(gè)實(shí)驗(yàn)室發(fā)現(xiàn)約100個(gè)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)DNACapAAAAAAAAAApri-miRNAmicroRNAExonIntronDNACapAAAAAAAAAAATGTGAmRNAProtein轉(zhuǎn)錄轉(zhuǎn)錄翻譯加工microRNA:由19-25個(gè)核苷酸組成的非編碼小分子RNA10BiogenesisandActionofmicroRNAs11MicroRNA-mRNAcomplexinterplayOnemiRNAtargetsmultiplemRNAsOnemRNAbymultiplemiRNAs12MicroRNAscanregulateabout~60%ofallmRNAs!MicroRNARegulationofCancerBiology13miR-125b,HepatologymiR-210HepatologymiR-423CarcinogenesismiR-151NatureCellBiologymiR-195,HepatologymiR-194,ScienceSignalingmiR-30d,HepatologymiR-124GastroentolgoymiR-199HepatologyMicroRNA領(lǐng)域值得關(guān)注的幾個(gè)問(wèn)題141.

microRNA生物合成及表達(dá)調(diào)控機(jī)制；2.microRNA在疾病進(jìn)程中的作用及調(diào)控網(wǎng)絡(luò)；（疾病模型）3.microRNA在生理、病理中的調(diào)節(jié)作用；（KO，TG）4.microRNA轉(zhuǎn)化醫(yī)學(xué)研究：血清診斷、預(yù)后判斷、分子分型、治療探索等Contents15

SmallRNAs(microRNAs);

Longnon-codingRNAs(lncRNAs);

CircularRNAs(circRNAs).LncRNADiscovery16LncRNA是一類轉(zhuǎn)錄本長(zhǎng)度超過(guò)200nt的RNA分子1980s-1990s:IndividuallncRNAs(eg,XistandH19)arediscoveredthroughtraditionalgenemappingapproaches;

Early2000s:LargescalecDNAsequencingleadstothediscoveryofasurprisingnumberoflncRNAtrancscripts;

2000s-Now:ThenumberofpredictedproteincodinggenesinhumangoesdownandthenumberofdetectedlncRNAtranscriptsincreases.GENECODEV21LncRNA分類和特征17根據(jù)基因組位置分為5類：

雙重活性：可結(jié)合蛋白與核酸（RNA,DNA）TrendsGenet.2014Oct;30(10):439-52.LncRNA具復(fù)雜的二級(jí)與高級(jí)結(jié)構(gòu)，決定其分子調(diào)控多樣性NatRevGenet.2014Jan;15(1):7-21.Sense,Antisense,Intronic,Intergenic,BidirectionalLncRNA功能和作用機(jī)制18ModelsofnuclearlncRNAfunctionModelsofcytoplasmiclncRNAfunctionNatRevGenet.2014Jan;15(1):7-21.mRNA穩(wěn)定性mRNA翻譯miRNAsponge蛋白穩(wěn)定性/修飾染色質(zhì)重塑表觀遺傳修飾基因轉(zhuǎn)錄可變剪接LncRNA具有重要分子調(diào)節(jié)功能，而不是“轉(zhuǎn)錄的噪音”19BiochimBiophysActa.2014;1839(11):1097-109.LncRNA與癌癥密切相關(guān)OncogenicandTumorsuppressivelncRNAs20HOXlincRNAsaresystematicallydysregulatedinbreastcancerThelincRNAHOTAIRisincreasedinexpressioninprimarybreasttumoursandmetastases

HOTAIRhasprognosticvalueformetastasisandsurvivalHOTAIRpromotesinvasionofbreastcarcinomacells.HOTAIRpromotesmetastasisofbreastcarcinomacells.ThemechanismofHOTAIRinbreastcancerprogressionLncRNA參考文獻(xiàn)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(guān)鍵詞：腫瘤類型+lncRNAContents28SmallRNAs(microRNAs);Longnon-codingRNAs(lncRNAs);

CircularRNAs(circRNAs).29CircularRNAWiluszandSharp,Science,2013ExonicCircularRNA(circRNAs,環(huán)狀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

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...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(fā)現(xiàn)（1976）拓展（1990s）

熱點(diǎn)（2010s）3031JuliaSalzmanSalzmanet.al.,PLoSone,201232Memczaketal.,Nature,2013Hansenetal.,Nature,2013NikolausRajewskyMCD,Berlin,GermanyJ?rgenKjemsAarhusUniversity,Denmark33BiogenesisofcircularRNADirectbacksplicingExonskippingJeckandSharpless,NatureBiotechnology,201434Liangetal.,GenesandDevelopment,2014Starkeetal.,CellReports,2014Ashwal-Flussetal.,MolecularCell,2014Ivanovetal.,CellReports,2015Connetal.,Cell,2015BiogenesisofcircularRNAZhangetal.,Cell,2014環(huán)狀RNA研究現(xiàn)狀及問(wèn)題一、鑒定（高通量測(cè)序／生物信息分析）二、生物合成和調(diào)控三、功能和機(jī)制四、意義RNA前體剪切加工的副產(chǎn)物？對(duì)細(xì)胞功能？機(jī)制？相對(duì)穩(wěn)定，是否作為疾病診療靶點(diǎn)？如何生成？!

出核？去向（代謝）？鑒定方法；組織、癌癥相關(guān)環(huán)狀RNA的存在情況？數(shù)量？表達(dá)？35RNA高通量測(cè)序鑒定環(huán)狀RNA36人類正常和腫瘤組織中存在大量的環(huán)狀RNA38環(huán)狀RNA具有組織特異性，在腦組織高表達(dá)NTNTallcancertissuesHCCtissues環(huán)狀RNA在腫瘤組織中存在差異表達(dá)環(huán)狀RNA在腫瘤組織中具有重要的臨床意義環(huán)狀RNA具有重要的功能環(huán)狀RNA作用機(jī)制？環(huán)狀RNA作用機(jī)制44SummaryandprospectmiRNA1990-00s～2000明確單一LncRNA1980-90s～30000待探索多樣復(fù)雜circRNA1970-90s～100000待探索未知

發(fā)現(xiàn)

數(shù)量

功能

機(jī)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