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

描述（由申请人提供）：微小RNA（miRNA）是基因组编码的小RNA（~ 21 nt），其以序列特异性方式与靶mRNA结合以调节其翻译和/或衰变。除了其在调节胆固醇和脂肪酸代谢中的作用之外，丰富的肝脏特异性microRNA miR-122还通过与HCV RNA的5'非编码区中的靶位点结合来刺激病毒复制，从而在丙型肝炎病毒（HCV）和相关肝细胞癌的发病机制中发挥核心作用。已经描述了用于拮抗miR-122功能的许多反义方法，其涉及重复高剂量施用修饰的寡核苷酸。该提议描述了一种使用序列特异性核酶来抑制miR-122的新方法。核酶的优点是它具有催化作用;一旦被引入细胞，单个分子将使多个拷贝的miRNA靶分子结合。我们的研究设计利用了M1 RNA的特性，M1 RNA是大肠杆菌RNase P的催化亚基。将指导序列共价连接到M1 RNA上会产生一种定制的核酶，该核酶选择性地使互补靶RNA失活。我们最近已经证明了这种方法在拟南芥中破坏miRNA功能的有用性。目标1将采用RNA工程和体外进化策略来开发一系列基于M1 RNA的核酶，这些核酶将有效地和选择性地降解miR-122。目的2将评估这些核酶在具有高水平miR-122的Huh-7肝癌细胞中针对miR-122的功效。为了控制核酶的水平，它将从四环素调节的启动子表达。建立核酶表达、miR-122水平和靶基因表达之间的定量关系将是这项工作的基石。RNA酶保护和qPCR测定将分别用于测量核酶和miR-122的水平。将使用具有来自阳离子氨基酸转运蛋白1（CAT-1）mRNA的3个miR-122结合位点的海肾荧光素酶报告基因、5种内源性靶mRNA的qPCR以及这些蛋白质中每种的蛋白质印迹法监测靶基因表达。miRNA微阵列将用于确定核酶靶向miR-122的特异性。最后，将通过用与let-7a互补的序列替换核酶中的miR-122指导序列并由此靶向let-7a来测试该方法的通用性。这项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R 01应用奠定基础，该应用将使用本文开发的工具直接靶向HCV。公共卫生相关性：丙型肝炎病毒（HCV）在肝细胞中的复制是由肝脏中最丰富的小非编码RNA microRNA-122（miR-122）的结合刺激的。该提案描述了一种使用催化RNA酶灭活miR-122的新方法。长期目标是将该策略应用于HCV感染的治疗。

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.