LET-7 MICRORNA REPRESSION OF STEM CELL COMPETITION AND CLONAL EXPANSION

 描述（由申请人提供）：在人结肠中，改变的肠上皮细胞的克隆扩增似乎先于明显的转化和肿瘤发生。小鼠模型表明，这是通过体细胞遗传和表观遗传改变赋予干细胞的竞争优势而发生的。我们的目标是确定Let-7丢失对肠上皮干细胞活性和克隆扩增的影响。我们已经发现Let-7在通过耗尽肠上皮中的Let-7 miRNA来调节肠中的干细胞特征和干细胞表型中的非常显著的作用。我们在小鼠模型中使用组成型LIN 28 B转基因和早期（胚胎晚期）Cre-Lox介导的MirLet 7 c2/MirLet 7 b簇缺失来消除Let-7 miRNA。这些小鼠还发展出高度渗透的肿瘤表型，在小肠中出现腺癌。然而，我们不知道12种不同Let-7基因中每一种的相对贡献，我们也不知道成年人的急剧丧失如何影响肠上皮的稳态。为了解决这些问题，我们有兴趣通过诱导方法靶向成人肠上皮中的Let-7 miRNA。由于Let-7 miRNA家族的大尺寸（8个簇中的12个基因，编码9个独特的Let-7 miRNA），可能需要灭活多个Let-7基因以观察可测量的表型。或者，特定的Let-7基因可能发挥独特的作用。我们提出了一种新的方法来解决这些困境。为了探索Let-7 miRNA在控制干细胞活性和克隆扩增中的单独和组合作用，我们将通过体细胞诱变靶向肠上皮中表达的所有簇。这将为Let-miRNA在限制干细胞活性和克隆扩增中的功能提供重要的见解。我们的目标也是在单个动物中开发Let-7突变的镶嵌模式，其中1至7个Let-7簇在单个动物的整个肠道中以各种组合突变。通过在类肠组织中的努力，我们还将确定通过慢病毒介导的9种不同Let-7 miRNA中的每一种表达对生长、增殖和集落形成潜力的相对影响。我们还将研究生长抑制因子Apc，Pten，Smad 4和Trp 53如何影响Let-7的表达，其中由其失活引起的克隆扩增可能部分取决于未能维持足够水平的Let-7 miRNA。

 DESCRIPTION (provided by applicant): Clonal expansion of altered intestinal epithelial cells appears to precede frank transformation and tumorigenesis in the human colon. Mouse models indicate that this occurs through competitive advantages conferred to stem cells via somatic genetic and epigenetic alterations. Our objective is to determine the impact of Let-7 loss on stem cell activity and clonal expansion in the intestinal epithelium. We have discovered a very salient role for Let-7 in the regulation of a stem cell signature and a stem cell phenotype in the intestine via depletion of Let-7 miRNAs in the intestine epithelium. We achieved this in mouse models using a constitutive LIN28B transgene and early (late embryonic) Cre-Lox mediated deletion of the MirLet7c2/MirLet7b cluster for depleting Let-7 miRNAs. These mice also develop a highly penetrant tumor phenotype with the appearance of adenocarcinomas in the small intestine. However, we do not know the relative contribution of each of the 12 different Let-7 genes, and we do not know how precipitous loss in the adult may affect homeostasis of the intestinal epithelium. To address these questions we are interested in targeting Let-7 miRNAs in the adult intestinal epithelium via an inducible approach. Due to the large size of the Let-7 miRNA family (12 genes in 8 clusters, encoding 9 unique Let-7 miRNAs) multiple Let-7 genes may need to be inactivated in order to see a measureable phenotype. Alternatively, specific Let-7 genes may play a unique role. We propose a novel approach to address each of these quandaries. To explore the individual and combinatorial role of Let-7 miRNAs in controlling stem cell activity and clonal expansion we will target all of the clusters expressed in the intestinal epithelium via somatic mutagenesis. This will provide significant insight into the function of Let- miRNAs in restricting stem cell activity and clonal expansion. Our goal is also to develop a mosaic pattern of Let-7 mutations in a single animal, where 1 to 7 Let-7 clusters are mutated in various combinations throughout the intestine of a single animal. Through efforts in enteroids, we will also determine the relative effects on growth, proliferation, and colony forming potential through lentiviral-mediated expression of each of the 9 different Let-7 miRNA species. We will also examine how the growth repressive factors Apc, Pten, Smad4, and Trp53 affect expression of Let-7, where clonal expansion caused by their inactivation may depend, in part, on a failure to maintain sufficient levels of Let-7 miRNAs.