Coronavirus modulation of cellular microRNAs in the liver: role in hepatitis

冠状病毒对肝脏细胞 microRNA 的调节：在肝炎中的作用

• 批准号: 8324597
• 负责人: SONIA NAVAS-MARTIN
• 金额: $ 19.31万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-29 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8324597
• 关键词: Acute; Acute Hepatitis; Address; Animals; Binding Sites; Biological; Biological Assay; Cell physiology; Cells; Complex; Computer Simulation; Coronavirus; Coronavirus Infections; DNA Viruses; Data; Disease; Disease Outcome; Elements; Endothelial Cells; Experimental Models; Functional RNA; Gene Expression; Genes; Genome; Health; Hepatic; Hepatitis; Hepatitis B; Hepatitis C; Hepatocyte; Human; Immune system; In Vitro; Infection; Inflammation; Inflammatory; Interferon Type II; Interferons; Intervention; Investigation; Knowledge; Kupffer Cells; Liver; Liver diseases; Luciferases; Mediating; Messenger RNA; MicroRNAs; Microarray Analysis; Modeling; Molecular; Molecular Profiling; Murine hepatitis virus; Mus; Nuclear; Outcome; Pathogenesis; Pattern; Platelet Factor 4; Play; Poly(A)+ RNA; RNA Viruses; Regulation; Regulatory T-Lymphocyte; Relative (related person); Reporter; Reverse Transcriptase Polymerase Chain Reaction; Role; Small RNA; System; Testing; Therapeutic; Time; Translational Repression; Translations; Tropism; Untranslated Regions; Validation; Viral; Viral Genes; Viral hepatitis; Virus; Virus Diseases; base; cellular targeting; cytokine; human coronavirus; in vivo; innovation; knock-down; liver function; mRNA Transcript Degradation; macrophage; man; novel; pathogen; positional cloning; promoter; prototype; respiratory; response; transcription factor; virus host interaction

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are small RNAs that regulate gene expression by translational repression and/or mRNA degradation. miRNAs may also up-regulate translation under certain circumstances. There is increasing evidence indicating that miRNAs play a major role in fundamental cellular processes, the regulation of the immune system, as well as in the onset and progression of many diseases. The role of miRNAs in virus-host interaction is just starting to be addressed. miRNAs have been found in some viruses and miRNAs from the host cell may play a role in regulating viral genes and even determine viral tropism. We have investigated whether coronaviruses (CoVs), a group of emerging animal and human RNA viruses, may have the ability to modulate the expression of some cellular miRNAs, which may determine disease outcome. Using a reverse genetic system to manipulate the CoV genome, we have previously characterized the molecular determinants of CoV-induced hepatitis, showed that the ability of murine coronaviruses to induce hepatitis is virus strain specific, and identified cellular targets in the liver (hepatocytes, endothelial cells, and the resident macrophage Kupffer cells). In Preliminary Studies, we have used miRNA microarray analysis and validation by real-time RTPCR to provide evidence of the differential expression of selected cellular miRNAs in macrophages upon in vitro infection with various strains of murine CoVs that induce different hepatitis outcome in the mouse (from minimal inflammation to fulminant hepatitis). Changes in the macrophage miRNAs profiles after murine CoVs infection were overlapping but distinct, and were observed early after infection in the absence of type I IFNs, IFN-?, and pro-inflammatory cytokine secretion. Therefore, we hypothesized that early alteration of the miRNAs expression profile in the CoV-infected mouse liver is a consequence of virus infection, and that strain-specific changes in miRNAs expression contribute to the differences in hepatitis outcome. Thus, we propose the following Specific Aims: (1) Using microarray analysis and real time RT-PCR, we will determine alterations in miRNA profiles in the mouse liver and in primary liver cells (hepatocytes and Kupffer cells) freshly isolated from infected mice, which are different between murine CoVs that differ in hepatitis outcome (MHV-A59, acute moderate hepatitis; and MHV-3, fulminant hepatitis); (2) Based upon results in Specific Aim 1, we will subsequently identify potential cellular miRNAs targets in 3'UTRs by in silico and functional approaches, focusing on those miRNAs whose expression is differentially modulated (up or down) between the viruses listed above and might be involved in fulminant hepatitis; and, (3) We will define the biological effects of knocking-down selected miRNAs (identified in the previous aims) in Kupffer and regulatory T cells and determine the role that those selected miRNAs may have in CoV-induced hepatitis outcome by specific in vivo miRNA-silencing using antagomirs.

描述(申请人提供)：microRNAs(MiRNAs)是通过翻译抑制和/或mRNA降解来调节基因表达的小RNA。在某些情况下，miRNAs也可能上调翻译。越来越多的证据表明，miRNAs在基本的细胞过程、免疫系统的调节以及许多疾病的发生和发展中发挥着重要作用。MiRNAs在病毒-宿主相互作用中的作用才刚刚开始得到解决。在一些病毒中已经发现了miRNAs，来自宿主细胞的miRNAs可能在调节病毒基因方面发挥作用，甚至决定病毒的嗜性。我们研究了冠状病毒(CoV)，一组新出现的动物和人类RNA病毒，是否有能力调节一些细胞miRNAs的表达，这可能决定疾病的结果。使用反向遗传系统操纵CoV基因组，我们以前已经表征了CoV诱导的肝炎的分子决定因素，表明小鼠冠状病毒诱导肝炎的能力是病毒株特异性的，并确定了肝脏中的细胞靶点(肝细胞、内皮细胞和常驻巨噬细胞Kupffer细胞)。在初步研究中，我们使用miRNA微阵列分析和实时RT-PCR验证来提供证据，证明在体外感染不同毒株的小鼠冠状病毒时，选定的细胞miRNAs在巨噬细胞中的差异表达，这些毒株会导致不同的小鼠肝炎结局(从轻微炎症到重型肝炎)。小鼠冠状病毒感染后巨噬细胞miRNAs谱的变化是重叠但明显的，并且在感染后早期观察到没有I型IFN、干扰素-β和促炎细胞因子的分泌。因此，我们假设CoV感染的小鼠肝脏中miRNAs表达谱的早期变化是病毒感染的结果，并且miRNAs表达的毒株特异性变化导致了肝炎结局的差异。因此，我们提出了以下具体目标：(1)利用基因芯片分析和实时RT-PCR，我们将确定从感染小鼠新鲜分离的小鼠肝脏和原代肝细胞(肝细胞和Kupffer细胞)中miRNA的变化，这在不同肝炎结局的小鼠冠状病毒(MHV-A59，急性中度肝炎；MHV-3，重型肝炎)中是不同的；(2)基于特定目标1的结果，我们随后将通过电子和功能方法确定3‘UTRs中潜在的细胞miRNAs靶点，重点是那些在上面列出的病毒之间表达差异调节(上调或下调)并可能参与暴发型肝炎的miRNAs；以及，(3)我们将定义在Kupffer和调节性T细胞中击倒选定的miRNAs(在先前的AIMS中确定的)的生物学效应，并通过使用Anagomir在体内通过特异性miRNA沉默来确定这些选定的miRNAs在CoV诱导的肝炎结局中可能扮演的角色。