Modulation neuroinflammation through interference of cooperative microRNA-RNA-binding protein interactions
Modulation neuroinflammation through interference of cooperative microRNA-RNA-binding protein interactions
批准号:
9300853
负责人:
JEFFREY R. BENDER
金额:
$16.75万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2016
资助国家:
美国
项目状态:
已结题
起止时间:
2016-06-17 至 2018-05-31
关键词:
3&apos Untranslated RegionsAddressAdhesionsAnimal ModelAnimalsAutoimmune DiseasesBindingBinding SitesBiological AssayCell AdhesionCellsChronicClinicalDataDemyelinationsDiseaseDisease modelElementsExperimental Autoimmune EncephalomyelitisGene DeletionGene ExpressionGenesGenetic TranslationGlobinGranulocyte-Macrophage Colony-Stimulating FactorHuR proteinHumanImmuneImmunizeIn VitroInflammatoryIntegrinsInterferonsInterleukin-17LeadLeukocytesLymphocyteMapsMessenger RNAMicroRNAsModelingMolecularMolecular AnalysisMultiple SclerosisMusMyelinNuclearOligonucleotidesPathogenesisPathogenicityPeptidesPlayPost-Transcriptional RegulationProductionProteinsProteolipidsPublishingRNARNA StabilityRNA-Binding ProteinsRecruitment ActivityRegulationRelapseReporterRoleSeverity of illnessSiteSpinal CordSystemT-LymphocyteTNF geneTestingTherapeuticTissuesTranscriptTranscriptional RegulationTransgenic OrganismsTranslational ResearchUntranslated RegionsWorkbasecytokineexperimental studyimmunopathologyin vivoinhibitor/antagonistmRNA Stabilitymigrationmouse modelnervous system disorderneuroinflammationneuropathologynew therapeutic targetoligodendrocyte-myelin glycoproteinpreventprotein expression
中文摘要
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。
英文摘要
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.
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