Catalytic inactivation of miRNA function by customized RNase P-based ribozymes

基于 RNase P 的定制核酶催化 miRNA 功能失活

• 批准号: 7641896
• 负责人: Venkat Gopalan
• 金额: $ 22.5万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7641896
• 关键词: 3' Untranslated Regions; Address; Animals; Antisense Oligonucleotides; Arabidopsis; Base Pairing; Binding; Binding Sites; Biological Assay; Biological Models; Biotechnology; Catalytic Domain; Catalytic RNA; Cationic Amino Acid Transporter 1; Cells; Cellular biology; Cholesterol; Cholesterol Homeostasis; Development; Dose; Effectiveness; Engineering; Enzymes; Escherichia coli; Evolution; Exhibits; Gene Expression; Gene Transfer; Genetic Translation; Goals; Hepatitis C; Hepatitis C virus; Hepatocyte; In Vitro; Liver; Malignant Neoplasms; Malignant neoplasm of liver; Measures; Messenger RNA; Metabolism; Methods; MicroRNAs; Modification; Molecular Profiling; Monitor; Oligonucleotides; Oncogenes; Pathogenesis; Pharmacologic Substance; Plants; Play; Point Mutation; Post-Transcriptional Regulation; Primary carcinoma of the liver cells; Property; Protein Binding; Proteins; RNA; RNA Biochemistry; RNase P; Renilla Luciferases; Reporter; Research; Research Design; Ribonucleases; Ribosomal Proteins; Role; Site; Small RNA; Specificity; Testing; Tetracyclines; Therapeutic Intervention; Translations; Untranslated RNA; Untranslated Regions; Viral; Virus; Virus Diseases; Virus Replication; Western Blotting; Work; base; cancer cell; fatty acid metabolism; insight; mRNA Transcript Degradation; novel strategies; promoter; public health relevance; single molecule; tool; tumor; viral RNA

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are genomically-encoded small RNAs (~ 21 nts) that bind to target mRNAs in a sequence-specific manner to regulate their translation and/or decay. In addition to its role in regulating cholesterol and fatty acid metabolism, the abundant liver-specific microRNA miR-122 also plays a central role in the pathogenesis of Hepatitis C virus (HCV) and associated hepatocellular carcinoma by stimulating viral replication through its binding to target sites in the 5' noncoding region of HCV RNA. A number of antisense approaches, which involve the repeated high-dose administration of modified oligonucleotides, have been described for antagonizing miR-122 function. This proposal describes a novel approach using a sequence-specific ribozyme to inactivate miR-122. The advantage of a ribozyme is that it is catalytic; once introduced into the cell, a single molecule will inactivate multiple copies of the miRNA target. Our research design exploits the properties of M1 RNA, the catalytic subunit of Escherichia coli RNase P. The covalent attachment of a guide sequence to M1 RNA generates a customized ribozyme that selectively inactivates the complementary target RNA. We have recently demonstrated the usefulness of this approach to disrupt miRNA function in Arabidopsis plants. Aim 1 will employ both RNA engineering and in vitro evolution strategies to develop an array of M1 RNA-based ribozymes that will effectively and selectively degrade miR-122. Aim 2 will evaluate the efficacy of these ribozymes against miR-122 in Huh-7 liver cancer cells, which have high levels of miR-122. To control the level of ribozyme, it will be expressed from a tetracycline-regulated promoter. Establishing the quantitative relationship between ribozyme expression, the levels of miR-122 and target gene expression will be a cornerstone of this work. RNase protection and qPCR assays will be used to measure the levels of ribozymes and miR-122, respectively. Target gene expression will be monitored using a Renilla luciferase reporter with three miR-122 binding sites from the cationic amino acid transporter 1 (CAT-1) mRNA, qPCR for five endogenous target mRNAs, and by Western blotting for each of these proteins. miRNA microarrays will be used to determine specificity of the ribozyme for targeting miR-122. Lastly, the generality of this approach will be tested by replacing the miR-122 guide sequence in the ribozyme with one complementary to let-7a and thereby targeting let-7a. The overall goal of this R21 proposal is to determine the feasibility of disrupting miR-122 function with a ribozyme, and to lay the groundwork for a subsequent R01 application that will use the tools developed here to directly target HCV. PUBLIC HEALTH RELEVANCE: The replication of Hepatitis C virus (HCV) in liver cells is stimulated by the binding of microRNA-122 (miR-122), the most abundant small noncoding RNA in liver. This proposal describes a new approach to inactivating miR-122 using a catalytic RNA enzyme. The long term goal is to apply this strategy to the treatment of HCV infection.

描述（由申请人提供）：microRNA（miRNA）是基因组编码的小RNA（〜21 nts），它们以序列特异性方式与靶MRNA结合，以调节其翻译和/或衰减。除了调节胆固醇和脂肪酸代谢中的作用外，丰富的肝脏特异性micrORNA miR-122在丙型肝炎病毒（HCV）的发病机理中也起着核心作用，以及相关的肝细胞癌通过与5'非coders noncoders s hcv in hcv in the 5'noncodegn ncod of toget intain of tose intage Repine刺激病毒复制。已经描述了许多反义方法，这些方法涉及重复的高剂量施用改良的寡核苷酸，以拮抗miR-122功能。该建议描述了一种使用序列特异性核酶使miR-122失活的新方法。核酶的优点是它是催化性的。一旦引入细胞，单个分子将使miRNA靶标的多个拷贝失活。我们的研究设计利用了M1 RNA的性质，M1 RNA的性能，大肠杆菌RNase的催化亚基P.导向序列与M1 RNA的共价附着会产生定制的核酶，从而选择性地失活互补靶标RNA。我们最近证明了这种方法在拟南芥植物中破坏miRNA功能的有用性。 AIM 1将同时采用RNA工程和体外进化策略来开发一系列基于M1 RNA的核酶，这些核酶将有效，有效地降解miR-122。 AIM 2将评估这些核酶对miR-122的疗效在HUH-7肝癌细胞中，这些肝癌细胞具有高水平的miR-122。为了控制核酶水平，它将从四环素调节的启动子中表达。建立核酶表达之间的定量关系，miR-122和靶基因表达的水平将是这项工作的基石。 RNase保护和QPCR测定将分别用于测量核酶和miR-122的水平。靶基因表达将使用来自阳离子氨基酸转运蛋白1（CAT-1）mRNA，QPCR，五个内源性靶标mRNA的肾荧光素酶报道器，并具有三个miR-122结合位点的肾上腺荧光素酶报告基因，并且每种蛋白质中的蛋白质都斑点。 miRNA微阵列将用于确定针对miR-122的核酶的特异性。最后，该方法的普遍性将通过用一种互补的let-7a替换为核酶中的miR-122指南序列，从而靶向let-7a。该R21提案的总体目标是确定用核酶破坏miR-122功能的可行性，并为随后的R01应用程序奠定基础，该应用将使用此处开发的工具直接针对HCV。公共卫生相关性：肝细胞中丙型肝炎病毒（HCV）的复制受到microRNA-122（miR-122）的结合，这是肝脏中最丰富的小型非编码RNA。该建议描述了一种使用催化RNA酶灭活miR-122的新方法。长期目标是将此策略应用于HCV感染的治疗。