The role of microRNAs in pancreas development

microRNA 在胰腺发育中的作用

• 批准号: 7312217
• 负责人: MARKUS STOFFEL
• 金额: $ 22.6万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7312217
• 关键词: RNA interference; biotechnology; cell line; developmental genetics; double stranded RNA; embryonic stem cell; functional /structural genomics; gene deletion mutation; gene targeting; genetic regulation; genetic susceptibility; genetically modified animals; glucose tolerance test; high throughput technology; human genetic material tag; insulin; insulin sensitivity /resistance; laboratory mouse; microRNAs; noninsulin dependent diabetes mellitus; pancreas; pancreatic islet function; pancreatic islets; postmortem; proteomics

MicroRNAs (miRNAs) are a recently discovered class of evolutionary conserved noncoding RNAs that exist as short inverted repeats in the genomes of invertebrates and vertebrates. Unlike other small RNAs, many miRNAs exhibit highly specific and restricted spatial and temporal expression. MicroRNAs are believed to act by basepairing with the RNA of the 3'-untranslated sequence regions (3'-UTRs) of one or multiple target genes, which leads to post-transcriptional repression. The function of most miRNAs is currently unknown but several studies in worms and flies indicate that they regulate essential biological functions such as developmental timing, cell proliferation, cell death fat metabolism and stress resistance. We have recently identified several novel miRNAs are expressed in pancreatic beta-cells. These miRNAs are highly conserved between human and mouse, their expression is pancreatic islet-specific, and overexpression and repression in pancreatic beta-cells leads to inhibition and stimulation of insulin secretion, respectively. In this application we propose a series of molecular genetic, biochemical and bioinformatic studies to elucidate the molecular function of islet miRNAs. In aim 1 we will examine the role of islet-specific miRNAs in pancreatic beta-cell function in vitro by overexpression and inhibition using 2'-O-methyl RNAs that have the complimentary sequence to miRNAs expressed in beta-cells. In aim 2 we will examine the function of RNA silencing during development of the pancreas by studying a lox-P modified dicer conditional allele in pancreatic beta -cells, ngn3- and pdx-1-positive cells. We will also study the specific biological role of two novel islet miRNAs by generating mutant mice by targeted deletion and homologous recombination in ES cells. The pancreatic phenotype of these mice will be studied during development and in adult life. In aim 3 we propose to identify and validate target genes of pancreatic islet miRNAs using biochemical, bioinformatic and proteomic approaches. In aim 4 we will test the hypothesis if genetic variation in islet-enriched miRNAs or their respective target sequences predisposes to the development of type 2 diabetes. In summary, this research proposal will i.) establish the molecular function of miRNAs in the developing and mature pancreas, ii.) contribute to our basic understanding of regulatory networks in differentiating beta-cells, and iii.) provide the community with genetic information that can be used to test if RNA silencing in beta-cells contributes to genetic susceptibility in type 2 diabetes.

microRNA（miRNA）是最近发现的一类进化保守的非编码RNA，它们是无脊椎动物和脊椎动物基因组中短反复重复序列而存在的。与其他小型RNA不同，许多miRNA表现出高度特异性和有限的空间和时间表达。据信microRNA通过基于一个或多个靶基因的3'-非翻译序列区域（3'-UTR）的RNA来起作用，从而导致转录后抑制。大多数miRNA的功能目前尚不清楚，但在蠕虫和苍蝇中进行了几项研究表明，它们调节了基本生物学功能，例如发育时机，细胞增殖，细胞死亡脂肪代谢和抗压力抗性。我们最近确定了几种新颖的miRNA在胰腺β细胞中表达。这些miRNA在人和小鼠之间是高度保守的，它们的表达是胰岛特异性的，并且 胰腺β细胞中的过表达和抑制分别导致胰岛素分泌的抑制和刺激。在此应用中，我们提出了一系列分子遗传，生化和生化研究，以阐明胰岛miRNA的分子功能。在AIM 1中，我们将通过使用2'-O-O-甲基RNA在体外进行胰岛特异性miRNA在体外的胰腺β细胞功能的作用，这些2'-O-甲基RNA具有在β细胞中表达的miRNA的互补序列。在AIM 2中，我们将通过研究胰腺β-细胞，NGN3-和PDX-1阳性细胞中的LOX-P修饰的dicer条件等位基因来检查RNA沉默在胰腺发育过程中的功能。我们还将通过通过靶向缺失和ES细胞同源重组产生突变小鼠来研究两个新型胰岛miRNA的特定生物学作用。这些小鼠的胰腺表型将在发育和成人生活中进行研究。在AIM 3中，我们建议使用生化，生化和蛋白质组学方法来识别和验证胰岛miRNA的靶基因。在AIM 4中，我们将检验富含胰岛的miRNA中的遗传变异或它们各自的目标序列，易于发展2型糖尿病的发展。 总而言之，该研究建议将i。）建立miRNA的分子功能 开发和成熟的胰腺，ii。）有助于我们对区分β细胞的调节网络的基本理解，以及III。）为社区提供遗传信息，可用于测试β细胞中的RNA沉默是否有助于2型2型糖尿病中的遗传易感性。