基因组与蛋白组学技术微小RNA及其在医学中的应用.ppt

微小RNA及其在医学中的应用,蔡俊超中山医学院微生物教研室,一、microRNA的研究历史二、microRNA的特点和作用机制三、microRNA与疾病及潜在的治疗作用四、microRNA研究的一般步骤和方法五、以一篇文章为例介绍一下研究的过程,基因组学,RNA组学,蛋白质组学,后基因组时代的中心法则,RNA加工,基因调节,DNA修复和重组,基因组的保持,基因组的表达,后基因组时代的基因调控：RNA调控,2019年的诺贝尔生理学奖获得者：,AndrewZ.FireCraigC.Mello,一、microRNA的研究历史,人类基因组中，蛋白质编码区只占1-2。其它98的基因组有什么功能呢？约24为内含子序列，这么冗长的内含子序列有什么生物学功能呢？,micoRNA(miRNA),是广泛存在于真核生物中的一组短小的、不编码蛋白质的RNA家族，它们是由19-23个核苷酸组成的单链RNA（3端可有12个碱基长度的变化）表达具有组织特异性和阶段特异性。即：在不同组织中表达有不同类型的miRNA,在生物发育的不同阶段里有不同的miRNA表达与靶mRNA3-UTR结合，序列特异性的在转录后水平调控基因的翻译表达，在生物发育、脂肪代谢、细胞的分化、增殖和凋亡等过程中发挥着重要作用,第一个被发现的miRNA(lin-4)由哈佛大学的VictorAmbros发现的（1993）第二个被发现的miRNA（let-7）由哈佛大学的博士后FrankSlack(Ruvkun实验室）发现的（2000),VictorAmbros,GaryRuvkun,微小RNA（microRNA,miRNA）的发现,1993,2000,2019,2019,2019,2019,2019,2019,2019,microRNA的研究历史,Dr.VictorAmbros,1993,2000,2019,2019,2019,2019,2019,2019,2019,1993,2000,2019,2019,2019,2019,2019,2019,2019,1993,2000,2019,2019,2019,2019,2019,2019,2019,2019年命名为microRNA,1993,2000,2019,2019,2019,2019,2019,2019,2019,2019年入选science年度十大科学发现之首,1993,2000,2019,2019,2019,2019,2019,2019,2019,ThefirstdemonstrationofalinkbetweenmiRNAgenesandcancer.,1993,2000,2019,2019,2019,2019,2019,2019,2019,miRNAcontrolssomeplantphenotype,1993,2000,2019,2019,2019,2019,2019,2019,2019,Thefirstquantitativereal-timePCRdevelopedformiRNAprofiling,designedfortheamplificationofprecursormolecules.,1993,2000,2019,2019,2019,2019,2019,2019,2019,ThefirststudytoreportonmiRNAprofilingusinganoligonucleotidemicrochip.,Thefirstpapertoexplore,byusinggenome-widemiRNAprofilingbymicroarray,thepotentialimportanceofmiRNAsinthediagnosisandprognosisofahumanmalignancy,1993,2000,2019,2019,2019,2019,2019,2019,2019,AnelegantstudythatshowsapathogeneticlinkbetweenmiRNAsandtargetoncogenes.,1993,2000,2019,2019,2019,2019,2019,2019,2019,ThefirstpapertoshowthatthederegulationofasinglemiRNAgenecanleadtocancer.,1993,2000,2019,2019,2019,2019,2019,2019,2019,通过生物信息的手段分析了microRNA在蛋白相互作用网络和转录因子-microRNA相互调控网络中的作用,1993,2000,2019,2019,2019,2019,2019,2019,2019,21-ntdsRNAstargetingselectedpromoterregionsofhumangenescausedlong-lastingandsequence-specificinductionoftargetedgenes,1993,2000,2019,2019,2019,2019,2019,2019,2019,Nature449,682-688(11October2019)|,1993,2000,2019,2019,2019,2019,2019,2019,2019,SangerRelease10.0contains5071hairpinprecursormiRNAs,expressing4922maturemiRNAproducts,inprimates,rodents,birds,fish,worms,flies,plantsandvirusesinAugust2019,近年来被鉴定的miRNAs越来越多至今超过17000miRNAs已被发现可以免费获得microrna.sanger.ac.uk/sequences/,二、microRNA的特点和作用机制,microRNA分布,miRNA名称与编号1)miRNA成熟体命名规则(以动物miRNA为例)miRNA成熟体简写成miR，再根据其物种名称，及被发现的先后顺序加上阿拉伯数字，如hsa-miR-122；高度同源的miRNA在数字后机上英文小写字母(a,b,c,)，如hsa-miR-34a，hsa-miR-34b，hsa-miR-34c等；由不同染色体上的DNA序列转录加工而成的具有相同成熟体序列的miRNA，则在后面机上阿拉伯数字以示区分，如hsa-miR-199a-1和hsa-miR-199a-2；通常一个miRNA前体长度大约为7080nt，很可能两个臂分别产生miRNA以前的做法是：表达水平较高的miRNA后面不加任何符号，而表达水平较低的miRNA后面加上*号，如rno-miR-9*。有时带“*”的miRNA就根本不出现。在miRBase19.0中则以“-5p”和“-3p”分别命名。如hsa-miR-26b-5p和hsa-miR-26b-3p，分别表明从hsa-mir-26b前体的5端臂和3端臂加工而来的。,/-OH,matureandprimarymicroRNA的结构,Pri-miRNA,DroshaCleavagesite,Mature,5,3,RNA诱导沉默复合体(RNA-inducedsilencingcomplex，RISC)组分Dicer酶：具有RNaseIII结构域,在RNAi的起始阶段负责催化siRNA的产生,在RISC装配过程中起稳定RISC中间体结构和功能的作用,Argonaute蛋白：是RISC中的核心蛋白,有PAZ和PIWI两个主要的结构域,前者为siRNA的传递提供结合位点,后者是RISC中的酶切割活性中心;siRNA是RISC完成特异性切割作用的向导siRNA/mRNA：,5endofthesmallRNA,28,knownastheseedregiondonothaveacomplexsecondarystructureandarelocatedinaccessibleregionsoftheRNA,microRNA识别靶位点,microRNA的加工成熟与分布,据体内外实验研究表明miRNA的生成需要两个步骤：,1)由长的内源性转录本（pri-miRNA)经Drosha酶作用生成70nt左右的miRNA前体(pre-miRNA)，该过程发生在细胞核2)将pre-miRNA经Dicer酶作用加工为成熟miRNA，该过程发生在细胞质中。,microRNA的加工成熟,microRNA是动、植物自身基因组编码形成的一类小分子,首先由基因组转录形成长链RNA分子pri-miRNA,约60%的microRNA为独立转录表达；约15%miRNA为成簇存在而共同转录；其余还有约25%的miRNA定位于功能基因内含子，随基因转录表达。,Pri-miRNA经双链RNA核酸酶Drosha酶作用，加工形成70-100nt长度的pre-miRNA,Pre-miRNA在Exportin5介导作用下转运出胞核至胞质中进行下一步加工,Pre-miRNA在胞质中经双链RNA核酸酶Dicer酶作用加工形成单链成熟miRNA分子,miRNA的加工成熟过程与RNAi技术中常用的siRNA有许多相似之处均经过Dicer酶对双链RNA的识别加工而形成单链RNA分子。,Thetargetspecificity,andprobablyalsothefunctionalefficiency,ofamiRNArequiresthatthematuremiRNAstrandfromthemiRNA:miRNA*duplexbeselectivelyincorporatedintotheRISCfortargetrecognition.ThemiRNA*strandisprobablydegradedrapidlyonitsexclusionfromtheRISC,astherecoveryrateofmiRNA*fromendogenoustissuesis100-foldlowerthanthatofmiRNAs.thestabilityofthe5endsofthetwoarmsofthemiRNA:miRNA*duplexisusuallydifferent,miRNAsisalmostalwaysderivedfromthestrandwiththelessstable5endcomparedwiththemiRNA*strand.,microRNA的作用机制,miRNA发挥作用过程,19-23nt的成熟miRNA形成后与其他蛋白共同组成RNA介导的沉默复合体（RISC）参与RNA干扰途径,miRNA形成RISC复合体后可与特定的靶mRNA结合，这种结合不要求严格的互补配对。结合后导致靶mRNA翻译的抑制,翻译抑制,5endofthesmallRNA,28,knownasthe“seedregion”donothaveacomplexsecondarystructureandarelocatedinaccessibleregionsoftheRNA,microRNA识别靶位点,microRNA作用机制,miRNPs=miRNA+RISC,微小RNA的作用特点,主要是在蛋白质水平调控端-个核苷酸高度保守，主要作用在靶mRNA的端非翻译区（unstranslatedregion,UTR）分完全和不完全配对两种方式，哺乳动物主要为不完全配对结合，影响mRNA的成熟、转运、稳定性或者在直接调控翻译是翻译抑制还是剪切mRNA，除配对因素外，还取决于RISC中组装元件的功能，如Arg蛋白,miRNA与siRNA的联系：,均为Dicer的产物长度均为22nt左右，5端是磷酸基，3端是羟基均需Argonaute家族蛋白的存在同为RISC的组分二者进化关系上可能的两种推论：,siRNA是miRNA的补充miRNA在进化过程中替代了siRNA,沉默机制有重叠,miRNA与siRNA的区别,三、microRNA与疾病及潜在的治疗作用,miRNA,发育增殖(干细胞),疾病,分化凋亡,VictorR.Ambros,秀丽线虫C.elegans,1.调控发育过程,miR-124a局限表达于神经组织miR-1局限表达于骨骼肌和心肌,青鳉,斑马鱼,小鼠,果蝇,miRNA的表达具有组织特异性,(Science2019),3种miRNA控制造血干细胞向淋巴细胞的分化过程,2.调控细胞分化,3.miRNAs与心血管疾病,（调控心肌前体细胞的增殖）,4、miRNA与病毒感染,在肝炎模型中发现的肿瘤相关miRNA,HCV,HBV,has-miR-191,Worsesurvivalinacutemyeloidleukemia,p53tumorsupressorpathway,has-miR-34,has-let-7a,rasandc-mycgenes,has-miR-133b,Sequamouscellcarcinoma,has-miR-142-5p,Hematopoieticspecific,Normal,5.微小RNA与肿瘤的关系,微小RNA调控癌基因表达,下调miRNA表达，促进了E2F1的表达,WesternBlotting,NorternBlotting,有些微小RNA可能是致癌基因,mir-1792高表达,增加mir-1719b的表达，加速了c-myc诱导的淋巴瘤形成,miRNAs与肿瘤的关系,miRNA与疾病关系的研究策略,哪些miRNA表达分离,克隆,测序,miRNA文库筛选,miRNA表达谱分析,哪些基因是miRNA的靶基因,哪些重要的miRNA影响某种生命活动,miRNA在什么时候、在哪里表达,功能分析,治疗,诊断,四、microRNA研究的一般步骤和方法,miRNAFunctionalAnalysisStrategyOverview,Culturedcells,Increasetargetproteinorreporterexpression,IntroduceprecursormiRNAs(Pre-miRTM),Reducetargetproteinorreporterexpression,TargetgeneIDGenefunctionCellularprocessPhenotype,Loss-of-functionexperiment,IntroducemiRNAinhibitors(Anti-miR),Culturedcells,Gain-of-functionexperiment,microRNA研究的一般步骤和方法,microRNA研究的一般步骤和方法,Real-timePCR,Real-timePCR,Microarray,DirectandsensitivemiRNAprofilingfromlow-inputtotalRNA.RNA13,151-159(2019),Microarray-based,high-throughputgeneexpressionprofilingofmicroRNAs.NatMethods1,155-161(2019).,miR-122regulationoflipidmetabolismrevealedbyinvivoantisensetargeting.CellMetab3,87-98(2019).,TherapeuticpotentialofmiRNAsinhumandisease,TheTherapeuticPotentialofmicroRNAs.ByAndreasGBaderandPaulLammersatMirnaTherapeutics,Inc,TargetingliverspecificmiR-122,80,TherapeuticpotentialofmiRNAsincancer,81,Nature449,682-688(11October2019),5以一篇文章介绍一下研究的过程,OutofatotalofeightselectedmiRNAs,three(miR-155,miR-9andmiR-10b)werefoundtobemarkedlyupregulatedinbreastcancercellswhencomparedwitheitherprimaryhumanmammaryepithelialcells(HMECs)orwiththespontaneouslyimmortalizedMCF-10Acells,theexpressionlevelofmiR-10bwas50-foldhigherincellsoftheMDA-MB-231line,whicharecapableofmetastasizing,thanincellsoftheMCF-7humanbreastcancerline,whichhavelittleifanymetastaticpowers.ThiscorrelationindicatedthatmiR-10bmightwellhaveacausalroleinbreastcancermetastasis.,5.1miR-10bcellmigrationandinvasioninvitro5.2miR-10btumourinvasioninvivo5.3miR-10bdistantmetastasisinvivo5.4howmiR-10bexpressionisregulated?5.5WhatisadirectandfunctionaltargetofmiR-10b?5.6miR-10bexpressioniselevatedinmetastaticbreasttumours,5.1miR-10bcellmigrationandinvasioninvitro,firstperformedinvitroloss-of-functionanalysesbysilencingthemiRNAswithantisenseoligonucleotidessuggestingthateachofthetransfectedantisenseRNAsachievedagreaterthan50%inhibitionoftheactionsofitscognatemiRNA.,AlthoughneitherofthesetwoantisenseRNAsaffectedthemotilityofMDA-MB-231cells,silencingofmiR-10bledtoamorethantenfoldreductionintheinvasivepropertiesofthesecells,Transwellmigrationassay,Matrigelinvasionassay,Trypanblueexclusionassay(台盼蓝拒染法),Thisreductionwasnotduetoimpairmentofcellviability,Takentogether,theseobservationssuggestedthatmiR-10bfunctionisrequiredforinvitroinvasivenessbutnotforviabilityormotilityofthesemetastaticcells.,miR-10boverexpression,clonedthegenomicsequenceofthehumanmir-10bgeneintoagreenfluorescentprotein(GFP)-expressing,murinestem-cellretrovirus(MSCV)-derivedvector1,TheseresultsindicatedthatoverexpressionofmiR-10bissufficienttopromotebothmigrationandinvasioninvitro.,5.1miR-10bcellmigrationandinvasioninvitro5.2miR-10btumourinvasioninvivo5.3miR-10bdistantmetastasisinvivo5.4howmiR-10bexpressionisregulated?5.5WhatisadirectandfunctionaltargetofmiR-10b?5.6miR-10bexpressioniselevatedinmetastaticbreasttumours,5.2miR-10btumourinvasioninvivo,implantedmiR-10b-transducedormock-infectedSUM149cellsintothemammaryfatpadsofNOD-SCIDmice(该品系鼠为非肥胖糖尿病/严重联合免疫缺陷鼠，不但成熟T、B细胞联合免疫缺陷，而且单核巨噬细胞、NK细胞功能下降，补体系统不健全，人类原代癌症和肿瘤很容易在其体内成癌症和肿瘤).,GFPexpressionwasmaintainedinthetumourcells,thecontrolSUM149tumourswerestrictlynoninvasive,asshownbytheirconfinementwithinfibroticcapsules(A,B).miR-10b-overexpressingSUM149tumoursdisplayedamassivedesmoplasticreaction,withislandsofepithelialcancercellsthathadinvadedthestroma(C,D).apparentmuscularandvascularinvasion(E,F),苏木素-伊红染色,Ki-67proliferationmarker,MECA-32endothelialcellmarker.,TodeterminewhethermiR-10bexpressionintheprimarytumourswouldalsoaffectcellproliferationandtumourangiogenesis,thedistribution,butnotthetotalnumber,ofKi-671cellsinthemiR-10b-overexpressingSUM149tumourswasdistinctfromthatseeninthecontroltumours,QuantificationofKi-67staining(percentageofKi-671carcinomacellsamongtotalcarcinomacells;e)andvessels(usingMECA-32-stainedsections;f)atthecentreandtheedgeoftheSUM149tumours.n53miceat6weeksafterimplantation.,ProminentintratumouralvesselsareassociatedwiththeinvasionfrontofmiR-10b-overexpressingtumoursasdemonstratedbyMECA-32stainingofprimarymammarytumoursformedbySUM149cellsinfectedwiththemiR-10b-expressingoremptyvector,5.1miR-10bcellmigrationandinvasioninvitro5.2miR-10btumourinvasioninvivo5.3miR-10bdistantmetastasisinvivo5.4howmiR-10bexpressionisregulated?5.5WhatisadirectandfunctionaltargetofmiR-10b?5.6miR-10bexpressioniselevatedinmetastaticbreasttumours,5.3miR-10bdistantmetastasisinvivo,MECA-32-andKi-67-stainedsectionsofaprimarymammarytumourformedbymiR-10btransducedSUM149cells,atweek6afterorthotopictransplantation.RedarrowsinpanelAindicatetumourcellswithinavessel,andblackarrowsinpanelDindicateendothelialcells.Magnification:panel