microRNA研究

,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,microRNA,Small molecular, Big world,microRNA,的发现与研究历史,microRNA,2002,年,science,十大科学发现之首,DNA,RNA,蛋白质,基因组学,RNA,组学,蛋白质组学,蛋白质编码基因数量,人：,30,000,果蝇和线虫：,12,000-14,000,酿酒酵母：,6,200,假单孢菌：,5,500,人和鼠的蛋白质编码基因,99%,是共同的。,人个体间单倍体基因组的碱基差异,300,万个，其中,1,万个（,0.3%,）出现在蛋白质编码基因中。,98%,转录产物是非编码蛋白,RNA,。,2,倍,2,倍,RNA,的分类,细胞核和胞液 线粒体 功能,核蛋白体,RNA rRNA mt rRNA,核蛋白体组成成分,信使,RNA mRNA,mt,mRNA,蛋白质合成模板,转运,RNA tRNA mt tRNA,转运氨基酸,不均一核,RNA hnRNA,成熟,mRNA,的前体,小核,RNA snRNA,参与,hnRNA,的剪接、转运,小胞浆,RNA scRNA/7SL-RNA,蛋白质内质网定位合成的,信号识别体的组成成分,非编,码,RNA (,ncRNA,),：,不被翻译成蛋白质的,RNA,，这些,RNA,不被翻译成蛋白质，但是参与蛋白质翻译过程。,Noncoding,RNAs,(,ncRNAs,) in,eukaryotes,Small RNA (,sRNA,) in,bacteria,Small nuclear,RNAs,(,snRNAs,),Small,nucleolar,RNAs,(,snoRNAs,),MicroRNA(miRNA,),Small temporal,RNAs(stRNAs,),Small interfering,RNAs(siRNAs,),RNA interference (,RNAi,),Guide,RNAs(gRNAs,),tRNA,/mRNA:,tmRNA,有些小,RNA,分子能直接调控某些基因的开关从而控制细胞的生长发育并决定细胞分化的组织类型,小,RNA,分子本身又包含了若干类,RNA,，根据小,RNA,的生成、结构和功能大约可分为以下三类：,miRNA,（,microRNA,),siRNA,(small interfering RNA),其他小,RNA,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,mRNA 3-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,miRNA,具有高度保守性，即各种,miRNA,都能在其他种系中找到同源体；,miRNA,独有的特征：其,5,端第一个碱基对,U,有强烈的倾向性，而对,G,却有抗性，但第二到第四个碱基缺乏,U,，一般来讲，除第四个碱基外，其他位置碱基通常都缺乏,C,miRNA,执行一定的生物学功能：,对与其互补的,mRNA,表达水平具有调节作用；,一些偏大的,miRNA,(27nt),可能参与了基因组的重组装,参与生物的发育与多种生理、病理过程,为什么,miRNAs,非常重要,?,microRNAs,是一类新的基因调控因子,某些,miRNAs,控制了动植物的发育,理解,miRNA,的功能将有助于现在流行的,siRNA,microRNAs,有可能成为新型的疾病标志物,microRNAs,可能具有重要临床应用价值,实验结果：,RNA,干扰基因沉默,1993,2000,2001,2002,2003,2004,2005,2006,2007,microRNA,的研究,历史,Dr. Victor,Ambros,1993,2000,2001,2002,2003,2004,2005,2006,2007,1993,2000,2001,2002,2003,2004,2005,2006,2007,1993,2000,2001,2002,2003,2004,2005,2006,2007,2001,年 命名为,microRNA,1993,2000,2001,2002,2003,2004,2005,2006,2007,2002,年 入选,science,年度十大科学发现之首,1993,2000,2001,2002,2003,2004,2005,2006,2007,The first demonstration of a link between,miRNA,genes and cancer.,1993,2000,2001,2002,2003,2004,2005,2006,2007,miRNA,controls some plant phenotype,1993,2000,2001,2002,2003,2004,2005,2006,2007,The first,quantitative real-time PCR,developed for,miRNA,profiling, designed for the amplification of precursor molecules.,1993,2000,2001,2002,2003,2004,2005,2006,2007,The first study to report on,miRNA,profiling using an,oligonucleotide,microchip.,The first paper to explore, by using genome-wide,miRNA,profiling by,microarray,the,potential importance of,miRNAs,in the diagnosis and prognosis of a human malignancy,1993,2000,2001,2002,2003,2004,2005,2006,2007,An elegant study that shows a,pathogenetic,link between,miRNAs,and target,oncogenes,.,1993,2000,2001,2002,2003,2004,2005,2006,2007,The first paper to show that the deregulation of a single,miRNA,gene can lead to cancer.,1993,2000,2001,2002,2003,2004,2005,2006,2007,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,1993,2000,2001,2002,2003,2004,2005,2006,2007,21-nt,dsRNAs,targeting selected promoter regions of human genes caused long-lastingand sequence-specific induction of targeted genes,1993,2000,2001,2002,2003,2004,2005,2006,2007,Nature,449, 682-688,(11 October 2007),|,1993,2000,2001,2002,2003,2004,2005,2006,2007,Sanger Release 10.0 contains 5071,hairpin precursor,miRNAs, expressing,4922 mature,miRNA,products, in,primates, rodents, birds, fish, worms ,flies, plants and viruses in August 2007,microRNAs,had been neglected for so many years because of their small size.,The underlying reason is: people never dream that small,RNAs,will have important biological roles.,Whats new about,miRNA,research in recent days?,1600,1200,No. of publications:,5,209,by 10/1/2008,1428,1307,940,828,800,484,400,163,0,2,6,36,1993,2001,2002,2003,2004,2005,2006,2007,2008,Year,No,. of,publications,microRNA,publications increased very rapidly,8186 papers/2010.07.19,7,By 2003:,145,genes/ 152 mature,miRNAs,(Sanger Release 3.0),By 2004:,192,genes/ 207 mature,miRNAs,(Sanger Release 5.1),By 2005:,281,genes/ 319 mature,miRNAs,(Sanger Release 7.1),By 2006:,411,genes/ 455 mature,miRNAs,(Sanger Release 9.0),By 2007:,541,genes/ 733 mature,miRNAs,(Sanger Release 10.1),By 2008:,695,genes/ 866 mature,miRNAs,(Sanger Release 12.0),Total number of,miRNA,genes predicted:,Whats new about,miRNA,research in recent days?,1,000,(,Landgraf,et al. 2007,Tuschl,Lab),25,000,(Miranda et al. 2006, IBM Bioinformatics Group),MicroRNA,publications increased very rapidly,How many,miRNAs,are there in human?,8,Whats new about,miRNA,research in recent days?,MicroRNA,publications increased very rapidly,How many,miRNAs,are there in human?,What regulates miRNAs,?,(He et al. 2007; Diederichs Viswanathan et al. 2008),New role of,miRNAs,as tumor suppressors,(He et al. 2007;,Mayr,et al. 2007),and tumor invasion and metastasis,(Ma et al. 2008),MicroRNAs,regulate stem cell self-renewal and differentiation,(Wang et al. 2007; Park et al. 2007; Yu et al. 2007; Yi et al. 2008;,Tay,et al. 2008),MicroRNAs,function in immune systems,(Li et al. 2007; Rodriguez et al. 2007; Thai et al. 2007),Viral,miRNAs,regulate (HIV or HSV-1) replication,(,Triboulet,et al. 2007;,Umbach,et al. 2008),MicroRNAs,can target 3-UTR, 5-UTR and coding regions,(Lytle et al. 2007;,Easow,et al. 2007;,Tay,et al. 2008),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,Overall, 98 of 186 (52.5%) of,miR,genes are in cancer-associated genomic regions or in fragile sites,microRNA,的分布,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,5end of the small RNA,28, known as the,“,seed region,”,do not have a complex,secondary structure and are located in accessible regions of the,RNA,microRNA,识别靶位点,microRNA,作用机制,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,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,。,miRNA,siRNA,产生,细胞内,RNA,的固有组分之一（正常）,RNAi,的活性形式，病毒感染和人工插入,dsRNA,之后诱导而产生（异常）,来源,内源转录本,转基因或病毒,RNA,（外源）,直接来源,发夹状,pre-,miRNA,长,dsRNA,结构,单链,双链，,3,端有,2,个非配对碱基，通常为,UU,互补性,不完全互补，存在错配现象,完全互补,对靶,RNA,特异性,相对较低，一个突变不影响,miRNA,的效应,较高，一个突变即引起,RNAi,沉默效应的改变,途径,miRNA,途径,RNAi,途径,对,RNA,的影响,在,RNA,代谢的各个层面进行调控,降解靶,mRNA,功能,调节内源基因的表达，在蛋白质合成水平发挥作用，与,mRNA,的稳定性无关,抑制转座子活性和病毒感染，在转录后水平发挥作用,影响,mRNA,的稳定性,miRNA,与,siRNA,的区别,microRNA,与肿瘤,Oncology, Bladder, Breast, Cervical, Colon, Leukemia, Lung, Pancreas, Prostate, Thyroid,Brain,Alzheimers, Multiple Sclerosis,Prion,Disease, Stroke, Miscellaneous, Atherosclerosis, Cardiac Hypertrophy, Diabetes, HIV/HPV Infection, Lupus, Cellular Process, Angiogenesis, Apoptosis, Cell Cycle, Neuronal Differentiation, Proliferation, Telomerase Activity, T-cell Activation, Various Cell Treatment,miRNA,Disease Analysis,microRNA,与肿瘤,E2F1,E2F1 Tsp1 CTGF,miR-1792,簇,miR-1792,簇位于,13q31,，其中包含了,7,个,microRNA: miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-20, miR-19b-1,和,miR-92-1,ODonnell,等发现,MYC,可以结合到,miR-17-92,簇所在基因,C13orf25,的第一个内含子上，并诱导,miR-17-92,簇的表达,与,p53,相关的,miRNA,2007-06-06,肿瘤有着独特的,miRNA,表达谱,microRNA,相关研究,microRNA,Expression Analysis,microRNA,Functional Analysis,miRNA,Expression Analysis,Strategy Overview,S,ample preparation,RNA isolation,cDNA,synthesis,Real-time PCR analysis,microRNA,cDNA,synthesis,stem-loop RT,poly(A,) tail RT,Real-time PCR workflow,microRNA,real-time PCR,SYBR Green,Taqman,probe,miRNA,Functional Analysis,Strategy Overview,Cultured cells,Increase target protein,or reporter expression,Introduce,precursor,miRNAs,(Pre-,miRTM,),Reduce target protein,or reporter expression,Target gene ID,Gene function,Cellular process,Phenotype,Loss-of-function experiment,Introduce,miRNA,inhibitors,(Anti-,miR,),Cultured cells,Gain-of-function experiment,http:/www.ebi.ac.uk/enright-srv/microcosm/cgi-bin/targets/v5/search.pl,http:/pictar.mdc-berlin.de/,http:/www.targetscan.org/,http:/www.microrna.org/microrna/home.do,http:/,diana.cslab.ece.ntua.gr,/,microRNA,序列数据库,http:/,www.mirbase.org,/,常用,microRNA,靶基因预测数据库,End,Mature,miRNA,“seed region”,How,miRNAs,findout,theis,targets?,Bases28,numered,fromthe5endof,miRNA, called,miRNA,seed, byusingseed,regionmiRNA,canbindtotargetsequence.,miRNA,bindingtopartiallycomplementarysitesinmRNA3,UTRs,is,consideredtobethe,halmark,ofanimal,miRNA,regulation.,Cell.2003Dec26;115(7):78798.,识别方式：,标准,5,优势性位点,种子结合,5,优势性位点,3,补偿性位点,