A role for microRNAs in global changes in gene expression induced by Leishmania

描述(申请人提供)：利什曼原虫。原生动物对它们入侵的宿主细胞有深远的影响。这些寄生虫通常居住在巨噬细胞内，巨噬细胞是一种能够杀死细胞内微生物的细胞类型。利什曼原虫不是屈服于杀菌机制，而是瘫痪宿主细胞的杀菌途径，将巨噬细胞从致命细胞变成细胞内的安全避风港，使寄生虫得以生存、复制并最终传播到邻近细胞。寄生虫对宿主的杀菌作用，其机制(S)尚不完全清楚。最近发现，大多数真核生物利用短的非编码RNA序列来全局调控各种基因的表达。事实上，长度为18-30bp的短RNA序列，称为microRNAs，对于调节大约30%的人类基因组的表达是至关重要的。虽然microRNAs可以触发mRNA的降解，但由microRNAs引起的许多变化都会在翻译水平上影响基因的表达。因此，通过对蛋白质的研究，微RNA的影响可能是明显的，但不一定是信使核糖核酸的丰度。支持这一建议的假设是，microRNAs是上游触发因素(S)，负责吞噬利什曼原虫查加西诱导的巨噬细胞基因表达的全球变化。此外，我们假设利什曼病操纵这些microRNAs作为一种手段，将巨噬细胞转换到与其自身生存相适应的环境中。这项建议的具体目标是：1.对人巨噬细胞吞噬查加西乳杆菌过程中和吞噬后诱导的microRNA表达的变化进行全球图谱分析。2.利用人类抗体微阵列，将microRNAs的变化与3‘UTRs中的潜在靶点以及与蛋白质表达的变化相关联。3.使用含有小鼠序列的微RNA微阵列芯片，在小鼠模型中检查感染小鼠的肝脏是否发生了相关的变化。这个为期两年的R21项目的近期目标将包括在巨噬细胞培养和体内实验中引入或击倒microRNAs，以努力系统地测试本文确定的microRNAs的生物学效应。最终目标包括扩展到人类系统，目的是开发新的治疗工具。公共卫生相关性：与公共卫生的相关性拉丁美洲内脏利什曼病是由原生动物利什曼原虫查加西引起的一种潜在的致命人类疾病。在这项研究中，我们将系统地研究微小RNA在利什曼原虫查加西感染过程中的作用，这是一种小的调节分子。这项研究的未来目标包括使用microRNAs开发治疗人类利什曼病的新靶点。

DESCRIPTION (provided by applicant): The Leishmania spp. protozoa have a profound effect on the host cell that they invade. These parasites reside intracellularly usually in macrophages, a cell type with the capacity to kill intracellular microbes. Rather than succumb to microbicidal mechanisms, Leishmania paralyze the microbicidal pathways of the host cell, changing the macrophage from a lethal cell to an intracellular safe haven which allows the parasite to survive, replicate and ultimately spread to neighboring cells. The mechanism(s) through which the parasite "paralyzes" the host microbicidal function is incompletely understood. It has recently come to light that most eukaryotic organisms utilize short noncoding RNA sequences to globally regulate expression of a wide variety of genes. Indeed short RNA sequences of 18-30 bp in length, called microRNAs, are critical for regulating expression of an estimated 30% of the human genome. Although microRNAs can trigger mRNA degradation, many changes induced by microRNAs affect gene expression at the level of translation. Thus the effects of microRNAs may be evident through studies of protein, but not necessarily mRNA abundance. The hypothesis underlying this proposal is that microRNAs are the upstream trigger(s) responsible for the global changes in macrophage gene expression induced by phagocytosis of Leishmania chagasi. Furthermore, we hypothesize that leishmania manipulate these microRNAs as a means of converting the macrophage in to an environment compatible with its own survival. The specific aims of this proposal are: 1. To perform a global profiling of changes in microRNA expression induced during and after phagocytosis of L. chagasi by human macrophages. 2. To correlate changes in microRNAs with potential targets in 3'UTRs, and with changes in protein expression using a human antibody microarray. 3. To examine in vivo in a mouse model whether correlated changes occur in the livers of infected mice, using microRNA microarray chips that contain murine sequences. Immediate future goals of this 2-year R21 project will include experimental introduction or knock-down of microRNAs in macrophage cultures and in vivo in an effort to systematically test the biological effects of microRNAs identified herein. Ultimate goals include the extension to a human system, with the intent of developing novel therapeutic tools. PUBLIC HEALTH RELEVANCE: RELEVANCE TO PUBLIC HEALTH Latin American visceral leishmaniasis is a potentially fatal human disease caused by the protozoan Leishmania chagasi. During this study we will systematically investigate the role of microRNAs, which are small regulatory molecules, in the progression of Leishmania chagasi infection. Future goals of this research include the use of microRNAs to develop novel targets for treatment of human leishmaniasis.