MicroRNA mediated inhibition of apoptosis in Coxiella burnetii infection

项目总结/摘要 Q热病原体贝氏柯克斯体抑制宿主细胞中的细胞凋亡（程序性细胞死亡）， 以便在稳定的细胞内区室中复制。柯克斯体分泌的效应蛋白进入 宿主细胞已经显示出抑制细胞凋亡，尽管它们作用机制并不完全 明白此外，目前还没有关于宿主microRNA的作用的信息。 在柯克斯体感染的细胞中，miRNAs可能在调节细胞凋亡中发挥作用。miRNAs是~22 nt的RNA 通过与靶点结合来调节人类细胞中的几种生物学过程，包括凋亡， mRNA和阻断翻译或引起靶降解。我们的长期目标是 了解非编码RNA如何调节柯克斯体-宿主相互作用。为了实现这一目标， 本申请的目的是鉴定在细胞凋亡过程中具有抑制细胞凋亡作用的miRNA， 柯克斯体感染。基于初步数据，揭示了与糖尿病相关的 我们的中心假设是，miRNAs在感染柯克斯体的巨噬细胞中发挥作用， 延缓细胞凋亡。本项目的目标将通过以下方式实现： 三个具体目标：（1）鉴定由柯克斯体感染诱导或抑制的miRNAs。使用 RNA-seq和qRT-PCR，我们将鉴定所有在柯克斯体中差异表达的miRNA。 感染的THP-1（人单核细胞/巨噬细胞）细胞。(2)确定抑制细胞凋亡的miRNAs 在Coxiella感染期间。对于在我们的初步研究和目标1中鉴定的miRNAs，我们将 测定它们抑制细胞凋亡的能力。(3)定义miR-148a在Coxiella感染过程中的作用。到 开始了解在分子水平上miRNAs如何促进抑制细胞凋亡在柯克斯体 感染后，我们将研究miR-148a的功能。选择这种miRNA是因为它 在我们的初步分析中，它在考克斯氏菌感染的细胞中显着下调，及其靶基因 （Bcl-2）在C.贝氏体发育和凋亡抑制。研究 这里提出的是创新，因为它将脱离现状，探讨一个全新的 宿主-病原体动力学领域：柯克斯体与人类细胞之间的RNA水平相互作用。这 这项工作意义重大，因为这将是第一项揭示miRNA在Coxiella感染中作用的研究。 感染，并根据我们的研究结果，新的药物，靶向miRNA可能 开发用于控制慢性Coxiella感染，这是难以治疗的，目前可用 抗生素我们的方法和结果也可以广泛应用于研究的功能， 在由其他细胞内病原体引起的感染期间的miRNA。

Project Summary/Abstract The Q fever pathogen Coxiella burnetii inhibits apoptosis (programmed cell death) in host cells in order to replicate in a stable intracellular compartment. Effector proteins secreted by Coxiella into host cell have been shown to inhibit apoptosis, although their mechanisms of action are not fully understood. Additionally, no information is currently available about the roles host microRNAs (miRNAs) potentially play in regulating apoptosis in Coxiella-infected cells. miRNAs are ~22 nt RNAs that regulate several biological processes in human cells, including apoptosis, by binding to target mRNAs and either blocking translation or causing target degradation. Our long-term goal is to understand how non-coding RNAs regulate Coxiella-host interactions. Towards attaining this goal, the objective of this application is to identify miRNAs that have a role in inhibiting apoptosis during Coxiella infection. Based on preliminary data that revealed potential functions for apoptosis-related miRNAs in Coxiella-infected macrophages, our central hypothesis is that miRNAs have a role in delaying apoptosis during Coxiella infection. The objective of this project will be accomplished by three specific aims: (1) Identify miRNAs that are induced or repressed by Coxiella infection. Using RNA-seq and qRT-PCR we will identify all miRNAs that are differentially expressed in Coxiella- infected THP-1 (human monocyte/macrophage) cells. (2) Determine miRNAs that inhibit apoptosis during Coxiella infection. For the miRNAs identified in our preliminary studies and in Aim 1, we will assay their ability to inhibit apoptosis. (3) Define the role of miR-148a during Coxiella infection. To begin to understand at a molecular level how miRNAs facilitate inhibition of apoptosis during Coxiella infection, we will interrogate the function of miR-148a. This miRNA was chosen because it was significantly down-regulated in Coxiella-infected cells in our preliminary analysis, and its target gene (Bcl-2) has critical roles in both C. burnetii development and apoptosis inhibition. The research proposed here is innovative, because it will depart from the status quo to investigate a whole new realm of host-pathogen dynamics: the RNA-level interaction between Coxiella and human cells. This work is significant because this will be the first study to uncover the roles of miRNAs during Coxiella infection, and based on our results, novel pharmaceutical agents that target miRNAs could potentially be developed to control chronic Coxiella infections, which are difficult to treat with currently available antibiotics. Our approach and results could also be broadly applied to studying the functions of miRNAs during infections caused by other intracellular pathogens.