医科大学精品课件：microRNA研究.ppt

1、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,基因组学,RNA组学,蛋白质组学,4,后基因组时代的中心法则,RNA processing,Gene regulation,DNA repair and recombination,基因组的保持,基因组的表达,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

2、.3%）出现在蛋白质编码基因中。 98%转录物是非编码蛋白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 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,编码蛋白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 RNA

3、s (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

4、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 (short 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

5、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

6、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,1993,2000,2001,200

7、2,2003,2004,2005,2006,2007,2002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

8、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 profil

9、ing 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

10、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-lasting an

11、d 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, b

12、irds, 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?,MicroRNA publicat

13、ions 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 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 miR

14、NAs (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

15、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 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

16、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-miR

17、NA,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。,The target specificity, and probably also the functional efficiency, of a miRNA requires that the mature miRNA strand from the miRNA:miRNA* dup

18、lex be selectively incorporated into the RISC for target recognition. The miRNA* strand is probably degraded rapidly on its exclusion from the RISC, as the recovery rate of miRNA* from endogenous tissues is 100-fold lower than that of miRNAs. the stability of the 5 ends of the two arms of the miRNA:

19、miRNA* duplex is usually different, miRNAs is almost always derived from the strand with the less stable 5 end compared with the miRNA* strand.,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

20、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,miRNPs=miRNA + RISC,miRNA与si

21、RNA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,microRNA与肿瘤, Oncology Bladder Breast Cervical Colon Leukemia Lung Pancreas Prostate Thyroid Brain Alzheimers Multiple Sclerosis Prion Disease Stroke, Misc

22、ellaneous 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,The let-7 family negatively regulat

23、es Ras,The miRNAs that are encoded by the let-7 family were the first group of oncomirs shown to regulate the expression of an oncogene, specifically the Ras genes. RAS原癌基因参与细胞的增殖和分化， 约有15-30%的肿瘤中含有RAS基因的突变,Ras家族有3个成员，即Nras、Kras、Hras分别含有9个、8个和3个let-7的靶位点，在HepG2细胞系中过表达let-7显著降低RAS蛋白的水平，在HeLa细胞系中转染let

24、-7的反义抑制分子，则会提高RAS蛋白的水平,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,He等将miR-1792簇中脊椎动物特异的部分miR-1719b 和c-myc在B细胞淋巴癌小鼠模型的造血干细胞中共表达，发现miR-1719b加快恶性淋巴瘤形成的速度，共表达miR-17

25、19b和c-myc的细胞比仅表达c-myc的细胞有更高的增殖速度和更低的死亡率 Dews等人发现miR-17-92簇可以抑制抗血管新生的因子Tsp1 和结缔组织生长因子CTGF，从而促进肿瘤中血管的新生，这些研究都表明miR-1792具有原癌基因的功能 而该簇中miR-17-5p和 miR-20a可以降低E2F1的水平， E2F1通过调控与DNA复制、细胞分裂和凋亡相关的基因控制细胞从G1期到S期， miR-17-92簇通过降低E2F1的水平又起到了抑癌基因的作用 miR-17-92簇是作为原癌基因还是抑癌基因发挥作用，要依赖于miRNA所在的组织、细胞类型以及胞内存在靶基因的种类,与p53相

26、关的miRNA,2007-06-06,肿瘤有着独特的miRNA表达谱,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 ex

27、periment,Introduce miRNA inhibitors (Anti-miR),Cultured cells,Gain-of-function experiment,End,Mature miRNA “seed region”,How miRNAs find out theis targets?,Bases 28, numered from the 5end of miRNA, called miRNA seed, by using seed regionmiRNA can bind to target sequence.,miRNA binding to partially complementary sites in mRNA 3 UTRs is considered to be the halmark of animal miRNA regulation. Cell. 2003 Dec 26;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,