Dicer as a repressor of antiviral response in embryonic stem cells

Dicer 作为胚胎干细胞抗病毒反应的阻遏物

• 批准号: 9516455
• 负责人: YAN-LIN GUO
• 金额: $ 44.4万
• 依托单位: UNIVERSITY OF SOUTHERN MISSISSIPPI
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9516455
• 关键词: Acute; Affect; Antiviral Agents; Antiviral Response; Apoptosis; Apoptotic; Biochemical; Biological Assay; Biological Models; Cell Death; Cell Proliferation; Cell physiology; Cell-Mediated Cytolysis; Cells; Chikungunya virus; DNA Transposable Elements; Defense Mechanisms; Development; Developmental Biology; Double-Stranded RNA; Elements; Embryonic Development; Ensure; Environmental Risk Factor; Enzymes; Event; Fluorescence; Genes; Genetic Transcription; Growth and Development function; Human; IRF3 gene; ISG15 gene; Immune; Immunology; Infection; Inflammation; Interferon Activation; Interferons; Knock-out; Label; Lead; Life Cycle Stages; Mammalian Cell; Mammals; Messenger RNA; MicroRNAs; Mus; Natural Immunity; Nuclear; Organism; Phenotype; Physiological; Plasmid Cloning Vector; Plasmids; Production; Property; RNA; RNA Interference; Regenerative Medicine; Reporter; Repression; Role; Sentinel; Series; Short Interspersed Nucleotide Elements; Signal Transduction; Somatic Cell; Stimulus; Stress; Structure; System; Testing; Time; Transcript; Uterus; Viral; Virus; Virus Diseases; West Nile viral infection; West Nile virus; base; cytotoxic; cytotoxicity; embryonic stem cell; expectation; experimental study; injured; insight; prevent; reconstitution; response; stable cell line; stem cell biology; transcription factor; viral RNA

PROJECT SUMMARY Early embryogenesis is the most vulnerable stage in the life cycle of mammals. Embryonic stem cells (ESCs) in the uterus have a dedicated task of rapid cell proliferation for the organismal development, but they can be adversely affected by many environmental factors, such as infection and inflammation. Currently, we know little about how ESCs cope with stress conditions. The interferon (IFN) system is the central part of antiviral innate immunity in differentiated somatic cells. We have recently demonstrated that ESCs are unable to express IFN, indicating that ESCs may not have a functional IFN-based antiviral mechanism. This finding raises important questions about the rationale for ESCs to not have such a critical defense mechanism and how do ESCs deal with viral infection. Using a synthetic dsRNA-based virus-free system, we have unexpectedly found that Dicer knockout ESCs (D-/-ESCs) seem to be able to express IFN and are highly sensitive to the increased cytotoxic effects of dsRNA. Similarly, B2RNA, a cellular RNA derived from the transcripts of transposable elements, showed remarkably similar effects to these caused by dsRNA. These findings challenge our view that the lack of IFN response is intrinsic in ESCs. Instead, our finding suggests that the basic machinery of the IFN system is functional in ESCs, but it may normally be repressed by Dicer. It is known that cellular RNA with dsRNA structures, derived from injured cells, misprocessed RNA and transcripts of transposable elements, can elicit antiviral responses that cause cellular damage similar to viral infection. We hypothesize that, by repressing IFN response, Dicer may act as a sentinel enzyme to prevent excessive antiviral responses that could potentially harm ESCs during early embryogenesis. Using mouse ESCs as a model system, we will test this hypothesis by: 1) using synthetic dsRNA as viral RNA mimics to determine the functionality of the major signaling events essential for the IFN system in D-/-ESCs (Aim 1), 2) using B2RNA as a physiologically relevant cellular RNA to investigate the activation of the IFN system and determine the role of Dicer in restricting B2RNA-induced cytotoxicity (Aim 2), and 3) reconstituting D-/-ESCs with ectopically expressed Dicer from both a plasmid vector and from synthetic Dicer-mRNA. The reconstituted ESCs will be used to validate the results obtained from dsRNA and B2RNA in D-/-ESCs. The successful completion of the proposed project is expected to provide significant insight into the unique immune properties of ESCs, specifically on how the IFN-based antiviral innate immunity is delicately controlled by Dicer to ensure normal growth and development at the early stage of organismal development.

项目摘要 早期胚胎发育是哺乳动物生命周期中最脆弱的阶段。胚胎干细胞（ESC） 子宫中的胚胎有一个专门的任务，即快速细胞增殖，以促进生物体的发育，但它们可以是 它受到许多环境因素的不利影响，如感染和炎症。目前，我们所知甚少 关于胚胎干细胞如何科普压力的。干扰素（IFN）系统是抗病毒天然免疫系统的核心部分， 分化体细胞的免疫力。我们最近已经证明ESC不能表达IFN， 表明ESC可能不具有基于IFN的功能性抗病毒机制。这一发现提出了重要的 关于ESC没有这种关键防御机制的理由以及ESC如何处理 病毒感染使用合成的基于dsRNA的无病毒系统，我们意外地发现Dicer 敲除的ESC（D-/-ESC）似乎能够表达IFN γ，并且对增加的细胞毒性非常敏感。 dsRNA的影响。类似地，B2 RNA，一种来自转座因子转录本的细胞RNA， 显示出与dsRNA引起的这些效应显著相似的效应。这些发现挑战了我们的观点， IFN应答的抑制是ESCs固有的。相反，我们的发现表明干扰素系统的基本机制 在ESC中有功能，但通常可能被Dicer抑制。已知具有dsRNA的细胞RNA 来自受损细胞的结构，错误加工的RNA和转座因子的转录本， 抗病毒反应导致细胞损伤，类似于病毒感染。我们假设通过抑制干扰素 Dicer可能作为一种哨兵酶，以防止过度的抗病毒反应， 在早期胚胎发育过程中伤害ESCs。我们将使用小鼠胚胎干细胞作为模型系统来验证这一假设 通过：1）使用合成的dsRNA作为病毒RNA模拟物以确定主要信号事件的功能性 对于D-/-ESCs中的IFN系统至关重要（目的1），2）使用B2 RNA作为生理相关的细胞RNA， 研究IFN系统的激活并确定Dicer在限制B2 RNA诱导的IFN-γ中的作用。 细胞毒性（目的2），和3）用异位表达的Dicer重建D-/-ESC，所述Dicer来自质粒载体 和合成的Dicer-mRNA。重构的ESC将用于验证从 在D-/-ESC中的dsRNA和B2 RNA。预计拟议项目的顺利完成将提供 对胚胎干细胞独特的免疫特性有重要见解，特别是关于基于干扰素的抗病毒药物如何 先天免疫由Dicer精细控制，以确保早期正常生长和发育 有机体发育阶段。