Roles for microRNA-122 in hepatitis C virus RNA amplification

microRNA-122 在丙型肝炎病毒 RNA 扩增中的作用

• 批准号: 8417691
• 负责人: PETER SARNOW
• 金额: $ 36.85万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-15 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8417691
• 关键词: Address; Adenosine; Affect; Anatomy; Animals; Antiviral Agents; Antiviral Therapy; Binding; Binding Sites; Biochemical; Cells; Cholesterol; Cholesterol Homeostasis; Chronic; Combined Modality Therapy; Complex; Confocal Microscopy; Cultured Cells; Dependence; Double-Stranded RNA; Down-Regulation; Drops; Elements; Enzymes; Eukaryotic Cell; Gene Expression; Genes; Genetic; Genome; Goals; Guanosine; HCV Vaccine; Healed; Health; Hepatitis C virus; Hepatocyte; Immune response; Individual; Infection; Interferons; Internal Ribosome Entry Site; Intervention; Lead; Life Cycle Stages; Liver; Liver diseases; Maintenance; Mediating; Messenger RNA; MicroRNAs; Modification; Monitor; Nucleotides; Occupations; Outcome Study; Pan Genus; Patients; Poly A; Polyadenylation; Primary carcinoma of the liver cells; Protein Isoforms; Proteins; RNA; RNA Degradation; RNA amplification; Recruitment Activity; Resistance; Ribavirin; Ribonucleases; Role; Science; Sequence Analysis; Serum; Site; Small Interfering RNA; Staging; Structure; Substrate Specificity; Testing; Transferase; Tretinoin; United States; Up-Regulation; Viral; Viral Genome; Viral Load result; Virus; cellular targeting; cholesterol biosynthesis; deep sequencing; effective therapy; fatty acid metabolism; genome-wide; healing; liver transplantation; locked nucleic acid; mimetics; mutant; novel; nuclease; public health relevance; research study; sensor; viral RNA

DESCRIPTION (provided by applicant): An estimated 170 million people worldwide and 4 million people in the United States are infected with hepatitis C virus (HCV). The majorities of patients do not resolve the infection and develop chronic infections that often lead to end-stage liver disease and hepatocellular carcinoma. Current treatment is limited to a combination therapy of ribavirin and interferon 1. This therapy is expensive and ineffective in 50% of infected individuals. Thus, there is an urgent need to identify viral or cellular molecules that can be used as novel targets in antiviral therapy. It was discovered that HCV binds two molecules of a liver-specific microRNA, miR-122, resulting in a novel, unprecedented upregulation of the viral genome. Sequestration of miR-122 in HCV-infected cultured cells or in livers of infected chimpanzees leads to a dramatic loss of infectious virus without emergence of resistant virus. Therefore, the dependence of HCV on miR-122 presents an Achilles heel of the virus that can be explored for antiviral intervention. This application proposes to study the roles for miR-122 in the viral life cycle and in cholesterol biosynthesis using a novel class of antisense molecules, locked nucleic acids (LNAs) that can easily be delivered to the liver in animals where it sequesters miR-122 in an inactive small duplex RNA. In particular, the first aim will characterize the RNA-RNA interactions in the miR-122/HCV complex, using genetic and biochemical approaches. Aim 2 will test the hypothesis that miR-122 protects the 5' end sequences of the HCV RNA from degradation by ribonucleases or RNA modification enzymes, or aids in the avoidance of activation of double-stranded RNA sensors such as the retinoic acid inducible gene I. These studies will be performed in specific and genome-wide siRNA-mediated gene knockdown experiments. Aim 3 proposes to examine roles for the known isoforms of miR-122 that contain extra 3' terminal adenosine or guanosine residues, on HCV RNA abundance. Deep sequencing analysis and gene knockdown of suspected nucleotidyl transferases will aid in this analysis. The final aim will characterize in detail the mechanism by which miR-122 regulates the expression of Insig1, the major negative regulator of cholesterol and fatty acid metabolism in the liver. In particular, the miR-122-mediated down-regulation of a distinct polyadenylation/cleavage site in a specific Insig1 isoform mRNAs will be examined. Overall, this application will address fundamental aspects about the functions of miR-122 in the HCV life cycle and cholesterol metabolism. The outcomes from these studies will detail novel mechanisms of gene expression mediated by microRNAs in eukaryotic cells and will point to new venues for antiviral therapies.

描述（由申请人提供）：全球估计有1.7亿人，在美国有400万人感染了丙型肝炎病毒（HCV）。大多数患者无法解决感染并发展出慢性感染，这些感染通常会导致末期肝病和肝细胞癌。当前的治疗仅限于利巴韦林和干扰素1的联合疗法。这种疗法在50％的感染者中昂贵且无效。因此，迫切需要鉴定可以用作抗病毒治疗的新靶标的病毒或细胞分子。已经发现HCV结合了肝特异性microRNA miR-122的两个分子，从而导致了一种新型的病毒基因组的前所未有的上调。 MiR-122在HCV感染的培养细胞或感染的黑猩猩肝脏中的静止会导致传染病的巨大丧失，而没有抗性病毒出现。因此，HCV对miR-122的依赖性提出了可以探索抗病毒干预的病毒的致命弱点。该应用建议使用新型的反义分子，锁定的核酸（LNA）来研究miR-122在病毒生命周期和胆固醇生物合成中的作用，这些分子可以轻松地将其在无效的小型二氧化双杆rna中在动物中轻松地传递到动物中。特别是，第一个目标将使用遗传和生化方法来表征miR-122/HCV复合物中RNA-RNA相互作用。 AIM 2将检验以下假设：miR-122保护HCV RNA的5'末端序列免受核糖核酸或RNA修饰酶降解的降解，或者有助于避免激活双链RNA传感器，例如视黄酸诱导的基因I。 AIM 3提出了检查MiR-122的已知同工型的作用，该miR-122含有额外的3'末端腺苷或鸟苷残基在HCV RNA丰度上的作用。可疑的核苷转移酶的深层测序分析和基因敲低将有助于该分析。最终目的将详细介绍miR-122调节Ingig1表达的机制，Indig1的表达是肝脏中胆固醇和脂肪酸代谢的主要负调节剂。特别是，将检查MiR-122介导的对特定Inig1同工型mRNA中不同聚腺苷酸化/裂解位点的下调。总体而言，该应用将解决有关miR-122在HCV生命周期和胆固醇代谢中功能的基本方面。这些研究的结果将详细介绍真核细胞中microRNA介导的基因表达的新机制，并将指向抗病毒药疗法的新场所。