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

描述（由申请人提供）：微小RNA（miRNA）是通过翻译抑制和/或mRNA降解调节基因表达的小RNA。在某些情况下，miRNAs也可以上调翻译。越来越多的证据表明，miRNAs在基本细胞过程、免疫系统的调节以及许多疾病的发生和发展中起着重要作用。miRNAs在病毒-宿主相互作用中的作用才刚刚开始得到解决。在一些病毒中发现了miRNAs，来自宿主细胞的miRNAs可能在调节病毒基因甚至决定病毒嗜性方面发挥作用。我们已经研究了冠状病毒（CoV），一组新兴的动物和人类RNA病毒，是否有能力调节一些细胞miRNA的表达，这可能决定疾病的结果。使用反向遗传系统来操纵CoV基因组，我们先前已经表征了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，我们使用了miRNA微阵列分析和实时RTPCR验证，以提供在体外感染各种鼠冠状病毒株后巨噬细胞中所选细胞miRNA差异表达的证据，这些病毒株在小鼠中诱导不同的肝炎结果（从轻微炎症到暴发性肝炎）。小鼠冠状病毒感染后巨噬细胞miRNAs谱的变化是重叠的，但不同，并且在缺乏I型IFN，IFN-β，和促炎细胞因子分泌。因此，我们假设，在冠状病毒感染的小鼠肝脏中的miRNA表达谱的早期改变是病毒感染的结果，并且miRNA表达的株特异性变化有助于肝炎结果的差异。因此，我们提出了以下具体的研究目的：（1）利用微阵列技术和真实的实时RT-PCR技术，检测小鼠肝脏和原代肝细胞中miRNA表达谱的变化（肝细胞和枯否细胞），其在肝炎结果不同的鼠冠状病毒之间是不同的（MHV-A59，急性中度肝炎; MHV-3，暴发性肝炎）;（2）基于特异性目的1的结果，我们随后将通过计算机模拟和功能方法鉴定3 'UTR中潜在的细胞miRNA靶点，重点关注那些表达受到差异调节的miRNAs（向上或向下）介于上面列出的病毒之间，可能与暴发性肝炎有关;并且，在本发明中，（3）我们将确定敲低选定的miRNAs的生物学效应在Kupffer和调节性T细胞中检测（在先前的目的中鉴定）的miRNA，并通过使用Escheromirs的特异性体内miRNA沉默来确定那些选择的miRNA在CoV诱导的肝炎结果中可能具有的作用。

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.