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Micro RNA Expression and Cancer

微小RNA表达与癌症

基本信息

批准号：
6895843
负责人：
THOMAS D. SCHMITTGEN
金额：
$ 13.46万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-05-20 至 2006-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6895843
关键词：
RNA carcinogenesis chronic lymphocytic leukemia clinical research complementary DNA fluorescent in situ hybridization gene expression high throughput technology human subject loss of heterozygosity method development microarray technology neoplasm /cancer genetics neoplastic process northern blottings polymerase chain reaction prostate neoplasms

项目摘要

Micro RNAs (miRNAs) are the smallest, functional, non-coding RNAs of plants and animals. To date over 200 miRNAs have been discovered across all multicellular phylogeny including C. elegans, Drosophila, plants, mice and humans. The mature miRNA is approximately 22 nt in length and is cleaved from partially duplexed precursors (termed pre-miRNA) by the ribonuclease III Dicer. Although over 100 human miRNAs have been discovered thus far, very little is known about their targets, levels of expression and function. Recently, two human miRNAs (miR15 and miR16) have been mapped to a region (13q14) that is commonly deleted in chronic lymphocytic leukemia (CLL). Northern blotting demonstrated that the expression of miR15 and miR16 was reduced in CLL patients with loss of heterozygosity (LOH) at 13q14. The purpose of this proposal is to develop sensitive assays to quantify miRNAs in clinical specimens. The long-term goal of this work is to investigate if a link exists between the expression of human miRNAs and the development of cancer. Since miRNAs are different from traditional RNAs they present distinct challenges as far as their quantification. Pre-miRNA exists as a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. We propose PCR methods to overcome these obstacles and permit sensitive, high-throughput quantification of this new class of RNAs. A real-time, quantitative PCR assay will be developed to screen the expression of all known 100+ human pre- and mature miRNAs. The real-time PCR data will be presented in a micro array format using a cluster algorithm to compare the relative ratios of the pre-miRNA. The assays will be tested and validated on cDNA from human tumor cell lines. The expression of miR15 and miR16 pre- and mature miRNAs will be measured in a pilot study of 40 CLL patients. The expression of both miRNAs will be correlated to the LOH at 13q14 in these patients. We will screen the expression of over 100 human pre-miRNAs in samples of normal, benign, primary and metastatic prostate tissue from 60 patients. We propose to develop sensitive, high-throughput methodologies to screen the expression of an entirely new class of genes that have only been discovered within the past two years. Since these are very small, noncoding RNAs, their expression is not possible using commercially available microarrays. Assays developed with this research may help to identify the function of a new class of genes in humans.

微小RNA（miRNAs）是植物和动物中最小的、功能性的、非编码RNA。迄今为止，在包括C.线虫、果蝇、植物、小鼠和人类。成熟的miRNA长度约为22 nt，并通过核糖核酸酶III Dicer从部分切割的前体（称为pre-miRNA）切割。尽管迄今为止已经发现了100多种人类miRNAs，但对它们的靶点、表达水平和功能知之甚少。最近，两种人类miRNAs（miR 15和miR 16）已被定位于慢性淋巴细胞白血病（CLL）中常见缺失的区域（13 q14）。北方杂交结果显示，在13 q14杂合性缺失（洛）的CLL患者中，miR 15和miR 16的表达降低。这样做的目的目前的一项建议是开发灵敏的测定方法来定量临床标本中的miRNA。这项工作的长期目标是研究人类miRNAs的表达与癌症的发展之间是否存在联系。由于miRNA不同于传统的RNA，因此就其定量而言，它们提出了不同的挑战。pre-miRNA以稳定的发夹形式存在，成熟的miRNA大致为标准PCR引物的大小。我们提出PCR方法来克服这些障碍，并允许敏感，高通量定量这类新的RNA。将开发实时定量PCR测定以筛选所有已知的100+人类前和成熟miRNA的表达。实时PCR数据将使用聚类算法以微阵列格式呈现，以比较pre-miRNA的相对比率。将对来自人肿瘤细胞系的cDNA进行检测和验证。将在40名CLL患者的初步研究中测量miR 15和miR 16前和成熟miRNA的表达。这两种miRNAs的表达与13 q14的洛缺失相关。我们将在来自60名患者的正常、良性、原发性和转移性前列腺组织样本中筛选超过100种人前体miRNA的表达。我们建议开发灵敏的高通量方法来筛选在过去两年中才发现的全新基因的表达。由于这些都是非常小的非编码RNA，它们的表达是不可能使用市售的微阵列。这项研究开发的检测方法可能有助于确定人类中一类新基因的功能。