Modulation neuroinflammation through interference of cooperative microRNA-RNA-binding protein interactions

通过干扰 microRNA-RNA 结合蛋白相互作用来调节神经炎症

基本信息

批准号：
9300853
负责人：
JEFFREY R. BENDER
金额：
$ 16.75万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-17 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9300853
关键词：
3&apos Untranslated Regions Address Adhesions Animal Model Animals Autoimmune Diseases Binding Binding Sites Biological Assay Cell Adhesion Cells Chronic Clinical Data Demyelinations Disease Disease model Elements Experimental Autoimmune Encephalomyelitis Gene Deletion Gene Expression Genes Genetic Translation Globin Granulocyte-Macrophage Colony-Stimulating Factor HuR protein Human Immune Immunize In Vitro Inflammatory Integrins Interferons Interleukin-17 Lead Leukocytes Lymphocyte Maps Messenger RNA MicroRNAs Modeling Molecular Molecular Analysis Multiple Sclerosis Mus Myelin Nuclear Oligonucleotides Pathogenesis Pathogenicity Peptides Play Post-Transcriptional Regulation Production Proteins Proteolipids Publishing RNA RNA Stability RNA-Binding Proteins Recruitment Activity Regulation Relapse Reporter Role Severity of illness Site Spinal Cord System T-Lymphocyte TNF gene Testing Therapeutic Tissues Transcript Transcriptional Regulation Transgenic Organisms Translational Research Untranslated Regions Work base cytokine experimental study immunopathology in vivo inhibitor/antagonist mRNA Stability migration mouse model nervous system disorder neuroinflammation neuropathology new therapeutic target oligodendrocyte-myelin glycoprotein prevent protein expression

项目摘要

The importance of Th17 cells, and their potent inflammatory cytokines (IL-17A and GM-CSF), in multiple sclerosis (MS) and other autoimmune diseases is established. In animal MS models, Th17 cells are recruited and localized to the CNS through 2 integrin LFA-1-dependent adhesion and transendothelial migration. Although transcriptional regulation of the IL-17A and GM-CSF genes has been well characterized, the mRNAs encoding these cytokines are highly labile and must be dynamically regulated to allow significant gene expression. We have demonstrated that T cell adhesion through 2 integrin engagement results in marked stabilization of mRNAs encoding TNF- and IFN-, through modulation and nuclear-to-cytosolic translocation of the RNA-binding protein (RBP) HuR. Our preliminary data support an equally remarkable extension of the IL-17A and GM-CSF transcript half-lives through an LFA-stimulated, HuR-dependent mechanism. When attempting to characterize potential competitive microRNA (miRNA)- HuR interactions on the IL-17A 3'- untranslated region (3'-UTR), we unexpectedly detected a cooperative, interdependent RNA- stabilizing interaction between miR-466l-3p and HuR. We mapped the miR-466l-3p target site within the IL-17A 3'-UTR. An oligonucleotide preventing this interaction (target site blocker [TSB]) inhibits LFA-1-induced, HuR- dependent IL-17A mRNA stabilization, and enhanced IL-17A production, in a cytokine-specific manner. We intend to define the same for GM-CSF, as its mRNA's 3'-UTR contains a highly conserved AU-rich element which includes 4 potential miR-466l-3p target sites. Our previously published and new data, and the pathogenic importance of IL-17A and GM-CSF in neuroinflammation, have led to our hypothesis, that leukocyte integrin engagement promotes Th17 cell IL-17A and GM-CSF expression via enhanced cooperative binding of HuR and miR-466l-3p to their 3'-UTRs, and that this potent pro-inflammatory switch is amenable to novel therapeutic targeting. Specific proposals now are to: (1) map the miR-466l-3p target site in the GM-CSF 3'-UTR, and generate an effective, specific TSB, using complementary molecular approaches including (a) MS2-TRAP 3'-UTR/miRNA pulldowns, and (b) pBBB  globin RNA reporter stability assays; and (2) determine the impact of selectively blocking miR-466l-3p's interaction with the IL-17A and GM- CSF transcripts on immunopathology in a chronic, myelin oligodendrocyte glycoprotein (MOG)-specific 2D2 transgenic EAE model, and a relapsing, remitting, proteolipid protein peptide (PLP)-immunized EAE model, evaluating EAE clinical scores, as well as CNS IL-17A and GM-CSF mRNA and protein levels. The novelty of this newly described cooperative miRNA-RBP interaction, and our ability to test inhibitors directed at this cooperativity in neuroinflammation disease models, makes this both a molecular and a highly translational exploratory R21 project. We hope this work directs more extensive efforts in posttranscriptional regulation of pathogenic cytokine expression, and defines a novel therapeutic targeting opportunity.

Th17 细胞及其强效炎症细胞因子（IL-17A 和 GM-CSF）在多种疾病中的重要性在动物 MS 模型中建立了 Th17 细胞。并通过 2 整合素 LFA-1 依赖性粘附和跨内皮迁移定位于 CNS。尽管 IL-17A 和 GM-CSF 基因的转录调控已得到很好的表征，但 mRNA 编码这些细胞因子的基因高度不稳定，必须动态调节以允许重要的基因我们已经证明，T 细胞通过 2 整合素接合产生显着的粘附作用。通过调节和核到胞质易位稳定编码 TNF-α 和 IFN-α 的 mRNA 我们的初步数据支持了 RNA 结合蛋白 (RBP) HuR 的同样显着的扩展。 IL-17A 和 GM-CSF 转录物半衰期通过 LFA 刺激、HuR 依赖性机制实现。尝试表征 IL-17A 3'- 上潜在的竞争性 microRNA (miRNA)-HuR 相互作用在非翻译区（3'-UTR）中，我们意外地发现了一个合作的、相互依赖的 RNA 稳定作用我们将 miR-466l-3p 与 HuR 之间的相互作用映射到 IL-17A 3'-UTR 内。阻止这种相互作用的寡核苷酸（靶点阻断剂 [TSB]）可抑制 LFA-1 诱导的 HuR- 我们以细胞因子特异性的方式依赖 IL-17A mRNA 稳定，并增强 IL-17A 的产生。打算对 GM-CSF 进行相同的定义，因为其 mRNA 的 3'-UTR 包含高度保守的富含 AU 的元件其中包括 4 个潜在的 miR-466l-3p 靶位点，以及我们之前发布的新数据。 IL-17A 和 GM-CSF 在神经炎症中的致病重要性导致了我们的假设：白细胞整合素结合促进 Th17 细胞 IL-17A 和 GM-CSF 表达 HuR 和 miR-466l-3p 与其 3'-UTR 协同结合，并且这种有效的促炎作用开关适合新的治疗靶向。现在的具体建议是：（1）绘制 miR-466l-3p 的图谱。 GM-CSF 3'-UTR 中的靶位点，并使用互补分子生成有效的、特异性的 TSB 方法包括 (a) MS2-TRAP 3'-UTR/miRNA Pulldowns 和 (b) pBBB  珠蛋白 RNA 报告稳定性 (2) 确定选择性阻断 miR-466l-3p 与 IL-17A 和 GM- 相互作用的影响 CSF 转录物对慢性髓磷脂少突胶质细胞糖蛋白 (MOG) 特异性 2D2 免疫病理学的影响转基因EAE模型，以及复发缓解型蛋白脂蛋白肽（PLP）免疫EAE模型，评估 EAE 临床评分，以及 CNS IL-17A 和 GM-CSF mRNA 和蛋白水平的新颖性。这种新描述的 miRNA-RBP 协同相互作用，以及我们测试针对此的抑制剂的能力在神经炎症疾病模型中的合作，使其成为一种分子和高度转化的方法我们希望这项工作能够在转录后调控方面发挥更广泛的作用。致病性细胞因子的表达，并定义了一种新的治疗靶向机会。