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

Th17细胞及其强大的炎性细胞因子(IL-17A和GM-CSF)在多发性硬化中的重要性 硬化症(MS)和其他自身免疫性疾病被确立。在动物多发性硬化模型中，Th17细胞被招募 并通过-2整合素LFA-1依赖的黏附和跨内皮细胞迁移定位于中枢神经系统。 尽管IL-17A和GM-CSF基因的转录调控已经被很好地描述，但mRNAs 编码这些细胞因子是高度不稳定的，必须动态调节以允许重要的基因 表情。我们已经证明，通过2整合素参与的T细胞黏附导致显著的 通过调节和核浆易位稳定编码肿瘤坏死因子-和干扰素-的mRNAs RNA结合蛋白(RBP)Hur。我们的初步数据支持同样值得注意的是 IL-17A和GM-CSF通过LFA刺激、HUR依赖的机制转录半衰期。什么时候 试图表征IL-17A 3‘-上潜在的竞争性microRNA(MiRNA)-HUR相互作用 非翻译区(3‘-UTR)，我们意外地检测到一个相互合作、相互依赖的RNA稳定 MiR-466l-3p与HUR的相互作用。我们将miR-466l-3p靶点定位在IL-17A 3‘-UTR内。 阻止这种相互作用的寡核苷酸(靶点阻断剂[TSB])抑制LFA-1诱导的Hur-1- 依赖IL-17A的mRNA稳定，并以细胞因子特异性的方式增加IL-17A的产生。我们 我打算为GM-CSF定义同样的内容，因为它的mRNA的3‘-UTR包含一个高度保守的富含AU的元件 其中包括4个潜在的miR-466l-3p靶点。我们之前发布的和新的数据，以及 IL-17A和GM-CSF在神经炎症中的致病作用导致了我们的假设，即 白细胞整合素参与增强Th17细胞IL-17A和GM-CSF的表达 HUR和miR-466l-3p与其3‘-UTRs的协同结合，以及这一强大的促炎作用 Switch适用于新的治疗靶向。现在的具体建议是：(1)映射miR-466l-3p 在GM-CSF 3‘-非编码区的靶点，并利用互补分子产生有效的、特异的TSB 方法包括(A)ms2-陷阱3‘-非编码区/miRNA下拉和(B)pBBB珠蛋白核糖核酸报告程序的稳定性 检测；以及(2)确定选择性阻断miR-466l-3p与IL-17A和GM-1相互作用的影响。 慢性髓鞘少突胶质细胞糖蛋白(MOG)特异性2D2的脑脊液免疫病理转录物 转基因EAE模型和复发、缓解、蛋白脂蛋白多肽(PLP)免疫的EAE模型， 检测EAE临床评分、中枢神经系统IL-17A、GM-CSF的mRNA和蛋白水平。的新奇之处 这种新描述的协作miRNA-RBP相互作用，以及我们测试针对这一点的抑制剂的能力 神经炎症性疾病模型中的协作性，使其既是分子的，又是高度翻译的 探索性R21项目。我们希望这项工作能更广泛地指导转录后调控。 致病细胞因子的表达，并定义了一个新的治疗靶向机会。

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.