《microRNA研究》PPT课件.ppt

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1、microRNASmall molecular,Big world,microRNA的发现与研究历史,microRNA2002年science十大科学发现之首,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。,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 m

2、RNA 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 eukaryotesSmall RNA(sRNA)in bacteria,Small nuclear RNAs(snRNAs)Small nucleolar RNAs(snoRNAs)Mi

3、croRNA(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

4、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

5、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siRNAmicroRNAs 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,1993,2000,2

6、001,2002,2003,2004,2005,2006,2007,1993,2000,2001,2002,2003,2004,2005,2006,2007,1993,2000,2001,2002,2003,2004,2005,2006,2007,2001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

7、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.,1993,2000,2001,2002,2003,200

8、4,2005,2006,2007,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,1993,2000,2001,2002,2003,2004,2005,2

9、006,2007,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

10、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)|,1993,2000,200

11、1,2002,2003,2004,2005,2006,2007,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 rea

12、son is:people never dream that small RNAs will have important biological roles.,Whats new about miRNA research in recent days?,microRNA publications increased very rapidly,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 gene

13、s/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(La

14、ndgraf et al.2007,Tuschl Lab)25,000(Miranda et al.2006,IBM Bioinformatics Group),MicroRNA publications increased very rapidlyHow many miRNAs are there in human?,8,Whats new about miRNA research in recent days?,MicroRNA publications increased very rapidlyHow many miRNAs are there in human?What regula

15、tes miRNAs?(He et al.2007;Diederichs 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；

16、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

17、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

18、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与siRNA的区别,microRNA与肿瘤,Oncology Bladder Breast Cervical Colon Leukemia Lung Pa

19、ncreas 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

20、Cell Treatment,miRNA Disease Analysis,microRNA与肿瘤,E2F1,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

21、AnalysismicroRNA Functional Analysis,miRNA Expression AnalysisStrategy Overview,microRNA cDNA synthesis,stem-loop RT,poly(A)tail RT,Real-time PCR workflow,microRNA real-time PCR,SYBR Green,Taqman probe,miRNA Functional AnalysisStrategy Overview,Cultured cells,Increase target proteinor reporter expre

22、ssion,Introduceprecursor miRNAs(Pre-miRTM),Reduce target proteinor reporter expression,Target gene IDGene functionCellular processPhenotype,Loss-of-function experiment,IntroducemiRNA inhibitors(Anti-miR),Cultured cells,Gain-of-function experiment,http:/,microRNA序列数据库,http:/,常用microRNA靶基因预测数据库,End,Ma

23、ture miRNA“seed region”,HowmiRNAsfindouttheistargets?,Bases28,numeredfromthe5endof miRNA,calledmiRNAseed,byusingseedregionmiRNAcanbindtotargetsequence.,miRNAbindingtopartiallycomplementarysitesinmRNA3 UTRsisconsideredtobethehalmarkofanimalmiRNAregulation.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,