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）的生命周期依赖于宿主脂质代谢，包括进入、复制和组装。肝脏中丰富的microRNA miR-122是病毒复制所需的宿主因子。这种前病毒机制部分是通过直接结合病毒基因组的5' UTR介导的。然而，直接结合并不能完全解释观察到的对病毒复制的影响。miR-122也被证明通过未知的机制调节胆固醇代谢。通过体外模型检测HCV病毒复制，我们证实胆固醇生物合成中间体不能挽救miR-122抑制的HCV复制。然而，LDL有效地恢复了复制。此外，我们证明载脂蛋白B100 （apoB100）本身从miR-122拮抗中拯救复制，并且单独强有力地促进病毒复制。反过来，rnai介导的apoB100的敲低减弱了miR122的前病毒作用，并且APOB基因双等位基因缺失的细胞系仅支持低水平的HCV病毒复制，并且miR-122过表达不能增强复制，而在野生型细胞中观察到的复制增加了2.5倍。这些数据表明，miR122发挥的前病毒作用部分是由apoB100介导的，并且apoB100是支持HCV复制的必要宿主因子。我们还证明，miR-122拮抗剂会导致apoB100蛋白表达显著降低，而不是mRNA水平。目前还不知道miR122功能和脂蛋白表达之间的机制联系。在本提案中，我们将(1)确定apoB100支持HCV复制的机制；(2)表征miR-122调控apoB100表达的机制。我们将通过评估miR-122对已知的对apoB100翻译后调控至关重要的蛋白质的影响来阐明连接miR-122和apoB100的调控途径。我们还将研究miR-122是否通过其对其他microrna的影响间接调节细胞胆固醇代谢，特别是microRNA-33，因为它已知参与胆固醇合成。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，并为慢性肝炎及其相关紊乱（包括脂肪变性）的发病机制提供见解。