microRNA研究课件.ppt

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： 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 2001年 命名为microRNA The first demonstration of a link between miRNA genes and cancer. The first quantitative real-time PCR developed for miRNA profiling, designed for the amplification of precursor molecules. An elegant study that shows a pathogenetic link between miRNAs and target oncogenes. The first paper to show that the deregulation of a single miRNA gene can lead to cancer. microRNA与肿瘤 The let-7 family negatively regulates 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-7的反义抑制分子，则会提高RAS蛋白的水平 の miR-17–92簇 miR-17–92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 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 experiment Introduce miRNA inhibitors (Anti-miR) Cultured cells Gain-of-function experiment Mature miRNA “seed region” How?miRNAs?find?out?theis?targets? Bases?2~8, numered?from?the?5’end?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):787‐98. 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 microRNA的加工成熟与分布 据体内外实验研究表明miR