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

• 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
• 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' 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

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细胞及其潜在炎症细胞因子（IL-17A和GM-CSF）的重要性， 硬化症（MS）和其他自身免疫性疾病已建立。在动物MS模型中，募集了Th17细胞 并通过2整联蛋白LFA-1依赖性的粘膜和跨内皮迁移定位于CNS。 尽管对IL-17a和GM-CSF基因的转录调节已经很好地表征了，但mRNA 编码这些细胞因子高度不稳定，必须动态调节以允许重要的基因 表达。我们已经证明，T细胞通过2整联蛋白参与的粘合剂导致标记 通过调节和核对偶然易位的编码TNF-和IFN-的mRNA稳定 RNA结合蛋白（RBP）HUR我们的初步数据支持了同样显着的扩展 IL-17A和GM-CSF转录本通过LFA刺激的Hur依赖性机制的半衰期。什么时候 试图表征潜在的竞争性microRNA（miRNA） - IL-17a 3'--的hur相互作用 未翻译的区域（3'-UTR），我们意外地检测到了一个合作，相互依存的RNA稳定化 miR-466L-3P与HUR之间的相互作用。我们在IL-17A 3'-UTR中绘制了miR-466L-3P目标位点。 防止这种相互作用的寡核苷酸（目标位点阻滞剂[TSB]）抑制LFA-1诱导的，hur- 依赖性IL-17A mRNA稳定，并以细胞因子特异性方式增强了IL-17A的产生。我们 打算为GM-CSF定义相同 其中包括4个潜在的miR-466L-3P目标位点。我们先前发布的新数据以及 IL-17A和GM-CSF在神经炎症中的致病意义已导致我们的假设，即 白细胞整联蛋白参与通过增强促进Th17细胞IL-17A和GM-CSF表达 HUR和miR-466L-3P的合作绑定与他们的3'-UTRS，并且这种有效的促炎性 开关适合新的治疗靶向。现在的具体建议是：（1）映射miR-466l-3p GM-CSF 3'-UTR中的目标位点，并使用完整的分子产生有效的特异性TSB 包括（a）ms2-trap 3'-utr/mirna pulldowns和（b）PBBBglobin RNA报告基因稳定性的方法 测定； （2）确定选择性阻断miR-466L-3P与IL-17A和GM-的相互作用的影响 慢性髓鞘少突胶质细胞（MOG）特异性2D2中的CSF成绩单 转基因EAE模型以及复发，恢复，蛋白质蛋白蛋白肽（PLP）免疫的EAE模型， 评估EAE临床评分，以及CNS IL-17A和GM-CSF mRNA和蛋白质水平。新颖的 这种新描述的合作miRNA-RBP相互作用以及我们测试针对此抑制剂的能力 神经炎症疾病模型中的合作性使它既是分子又是高度翻译的 探索性R21项目。我们希望这项工作将在转录后监管方面提供更广泛的努力 致病性细胞因子表达，并定义了一种新型的治疗靶向机会。