Characterization of microRNA-lipid-HCV interactions

microRNA-脂质-HCV 相互作用的表征

• 批准号: 8453061
• 负责人: ESPERANCE ANNE KREEK SCHAEFER
• 金额: $ 5.94万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8453061
• 关键词: 5' Untranslated Regions; Accounting; Animal Model; Apolipoproteins; Apolipoproteins B; Attenuated; Binding; Calnexin; Cell Line; Cells; Cholesterol; Cholesterol Homeostasis; Chronic Hepatitis; Chronic Hepatitis C; Cirrhosis; Complex; Data; Dependence; Diet; Dyslipidemias; Fatty Liver; Fatty acid glycerol esters; Functional RNA; GRP94; Gene Expression; Genes; Goals; Heat-Shock Proteins 70; Hepatitis C; Hepatitis C virus; Integration Host Factors; Interferons; Internet; Left; Link; Lipids; Lipoproteins; Liver; Liver diseases; Long-Term Effects; Low-Density Lipoproteins; Mediating; Messenger RNA; MicroRNAs; Mus; Pathogenesis; Population; Post-Translational Regulation; Primary carcinoma of the liver cells; Proteins; RNA Interference; RNA Viruses; Regulatory Pathway; Research; Resistance development; Serum; Staging; Translational Repression; Viral; Viral Genome; Viremia; Virus; Virus Diseases; Virus Replication; calreticulin; cell type; feeding; in vitro Model; insight; lipid metabolism; liver transplantation; mRNA Transcript Degradation; microsomal triglyceride transfer protein; overexpression; public health relevance; viral RNA; viral resistance

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) depends on host lipid metabolism for its lifecycle, including entry, replication and assembly. The liver-abundant microRNA miR-122 is a host factor required for viral replication. This pro-viral mechanism is in part mediated by direct binding to the 5' UTR of the viral genome. However, direct binding does not completely account for the observed effects on viral replication. miR-122 has also been demonstrated to regulate cholesterol metabolism through unknown mechanisms. Using an in vitro model examining HCV viral replication, we confirmed that cholesterol biosynthetic intermediates did not rescue miR-122- suppressed HCV replication. However, LDL effectively restored replication. Furthermore, we demonstrated that apolipoprotein B100 (apoB100) itself rescued replication from miR-122 antagonism, and alone robustly promotes viral replication. In turn, RNAi-mediated knockdown of apoB100 attenuated the proviral effect of miR122, and cell lines with biallelic deletion of the APOB gene support only low levels of viral replication with HCV, and miR-122 overexpression fails to enhance replication, compared to a 2.5-fold increase observed in the wild type cells. These data indicate that miR122 exerts a pro-viral effect that is mediated in part by apoB100, and that apoB100 is a necessary host factor to support HCV replication. We have additionally demonstrated that miR-122 antagonism leads to a significant reduction in apoB100 expression at the protein, but not mRNA level. There is not presently a known mechanistic link between miR122 function and lipoprotein expression. In this proposal, we will (1) determine the mechanism(s) by which apoB100 supports HCV replication; and (2) characterize the mechanisms by which miR-122 regulates expression of apoB100. We will clarify the regulatory pathways linking miR-122 and apoB100 by assessing the impact of miR-122 on proteins known to be critical for post-translational regulation of ApoB100. We will also examine whether miR-122 indirectly regulates cellular cholesterol metabolism through its impact on other microRNAs, and microRNA-33, in particular, given its known involvement in cholesterol synthesis. Taken together, these studies will serve to clarify the relationship between microRNA-122, HCV, and lipid metabolism and provide insights into the pathogenesis of chronic hepatitis and its related derangements, including steatosis.

描述（由申请人提供）：丙型肝炎病毒（HCV）取决于宿主脂质代谢的生命周期，包括进入，复制和组装。肝丰富的microRNA miR-122是病毒复制所需的宿主因子。这种临床机制部分是由直接结合与病毒基因组的5'UTR介导的。但是，直接结合并不能完全解释观察到的对病毒复制的影响。也已证明miR-122通过未知机制来调节胆固醇代谢。使用检查HCV病毒复制的体外模型，我们确认胆固醇生物合成中间体未挽救miR-122-抑制HCV复制。但是，LDL有效地恢复了复制。此外，我们证明了载脂蛋白B100（APOB100）本身从miR-122拮抗作用中拯救了复制，并且单独促进病毒复制。反过来，RNAi介导的APOB100的敲低抑制了MiR122的前病毒作用，而APOB基因的双重缺失的细胞系仅支持HCV的病毒复制水平较低，而MiR-122的过表达不足以增强复制，而与2.5倍相比，在野生型细胞中观察到了2.5倍。这些数据表明MiR122发挥了促病毒作用，该效应部分由APOB100介导，而APOB100是支持HCV复制的必要宿主因素。我们还证明，miR-122拮抗作用会导致蛋白质上的APOB100表达显着降低，但没有mRNA水平。目前，miR122功能和脂蛋白表达之间没有已知的机械联系。在此提案中，我们将（1）确定APOB100支持HCV复制的机制； （2）表征miR-122调节APOB100表达的机制。我们将通过评估miR-122对已知对APOB100的翻译后调节至关重要的蛋白质的影响来阐明连接miR-122和apoB100的调节途径。我们还将检查miR-122是否通过其对其他microRNA的影响和MicroRNA-33（尤其是）已知参与胆固醇合成来调节细胞胆固醇代谢。综上所述，这些研究将有助于阐明microRNA-122，HCV和脂质代谢之间的关系，并提供有关慢性肝炎及其相关危险（包括脂肪变性）的发病机理的见解。