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

描述(申请人提供)：microRNAs(MiRNAs)是基因组编码的小RNAs(~21nts)，它以序列特异性的方式与靶mRNAs结合，调节其翻译和/或衰退。除了调节胆固醇和脂肪酸代谢的作用外，丰富的肝脏特异性microRNA miR-122还通过与丙型肝炎病毒RNA 5‘非编码区的靶点结合来刺激病毒复制，从而在丙型肝炎病毒及其相关肝细胞癌的发病机制中发挥核心作用。已有许多反义方法被描述为拮抗miR-122功能，其中包括重复高剂量给予修饰的寡核苷酸。这项建议描述了一种使用序列特异性核酶来灭活miR-122的新方法。核酶的优点是它是催化的；一旦被引入细胞，单个分子就会使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是大肠杆菌RNaseP的催化亚单位。引导序列与M1 RNA的共价结合产生了一种定制的核酶，它选择性地灭活互补的靶RNA。我们最近已经证明了这种方法在扰乱拟南芥植物miRNA功能方面的有效性。AIM 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，这些核酶将有效和选择性地降解miR-122。目的2将评估这些核酶在Huh-7肝癌细胞中对miR-122的疗效，Huh-7肝癌细胞中miR-122水平较高。为了控制核酶的水平，它将从四环素调控的启动子中表达。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。核糖核酶保护法和qPCR法将分别用于检测核酶和miR-122的水平。目标基因的表达将使用带有阳离子氨基酸转运体1(CAT-1)mRNA3个miR-122结合位点的Renilla荧光素酶报告，5个内源性靶mRNAs的qPCR，以及这些蛋白的Western blotting来监测。MiRNA微阵列将用于确定针对miR-122的核酶的特异性。最后，将通过将核酶中的miR-122引导序列替换为与let-7a互补的序列从而靶向let-7a来测试该方法的一般性。R21提案的总体目标是确定用核酶干扰miR-122功能的可行性，并为后续的R01应用奠定基础，该应用将使用这里开发的工具直接靶向丙型肝炎病毒。与公共卫生相关：丙型肝炎病毒(HCV)在肝细胞中的复制受到microRNA-122(miR-122)的结合刺激，microRNA-122是肝脏中含量最丰富的非编码小RNA。这项建议描述了一种使用催化RNA酶灭活miR-122的新方法。长期目标是将这一策略应用于丙型肝炎病毒感染的治疗。

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.