In vivo studies of megakaryocyte microRNAs regulating platelet number and integrin activation

巨核细胞 microRNA 调节血小板数量和整合素激活的体内研究

基本信息

批准号：
9922374
负责人：
PAUL F. BRAY
金额：
$ 61.21万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-03-15 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9922374
关键词：
Abbreviations Ablation Affect Binding Binding Sites Biochemical Bioinformatics Biological Assay Blood Coagulation Disorders Blood Platelets Bone Marrow Bone Marrow Diseases Cardiovascular Diseases Cell Differentiation process Cells Chronic Myeloid Leukemia Clinical Collagen Collagen Receptors Communities Computational algorithm Data Data Set Defect Disease Drug Targeting Functional disorder Gene Expression Genes Genetic Genetic Transcription Genetic Translation Goals Health Hematology Hematopoiesis Hematopoietic stem cells Hemorrhagic Thrombocythemia High-Throughput Nucleotide Sequencing Human In Vitro Inflammation Inflammatory Integrins Interferon-alpha Interferons Knock-out Knowledge Lasers Mediating Megakaryocytes Messenger RNA MicroRNAs Molecular Molecular Genetics Mus Myeloproliferative disease Outcome Pathogenesis Pathogenicity Patients Peptides Platelet Count measurement Platelet Transfusion Play Polycythemia Vera Process Production Regulation Repression Research Role Seeds Signal Transduction Stress Testing Thrombocytopenia Thrombus Time Translations Umbilical Cord Blood base cohort crosslink crosslinking and immunoprecipitation sequencing cytokine drug development effective therapy genome wide association study genome-wide analysis hematopoietic differentiation in silico in vivo insight knock-down locked nucleic acid mRNA Expression mRNA Transcript Degradation male health novel overexpression parenteral administration platelet function prediction algorithm response stress disorder therapeutic miRNA therapeutic target thrombocytosis transcriptome transcriptome sequencing

项目摘要

Summary/Abstract Current knowledge about megakaryocyte (MK) gene expression has focused largely on transcription, whereas translational processes are relatively poorly understood in the healthy state, and even less is known in disease states. Dysregulated microRNAs (miRs) play a critical role in inflammation and the pathogenesis of several human bone marrow diseases, and over-expression and knockout studies in mice have identified miRs that induce myeloproliferative disorders (MPDs). Nevertheless, the role of miRs in MK gene expression in hu- man health or disease remains poorly understood. This research will use in vivo, in vitro, biochemical and bio- informatic approaches to study miR regulation of normal and stress (IFN)-induced MK gene expression. In our preliminary data, we identified miRs strongly associated with platelet count and function in 154 healthy do- nors. Knockdown and over-expression studies showed miR-125a-5p regulates proplatelet formation (PPF) and miR-15a-5p regulates GPVI-induced MK integrin activation in cultured MKs, and that both miRs are pre- dicted to target genes expressed in primary human bone marrow MKs. We hypothesize that these miRs regu- late in vivo megakaryocytopoiesis (MKpoiesis) and platelet production and function. Aim 1 will test this hy- pothesis in vivo using MK-specific ablation of miR-125a and miR-15a, as well as parenteral administration of anti-miRs, using assays of platelet production, Mkpoiesis, and GPVI-mediated platelet reactivity, thrombus formation and signaling. To understand how miRs function it is critical to know their mRNA targets. Until re- cently, the approaches for identifying miR targets have been based on computer algorithms with high false positive and false negative prediction rates, such that additional biochemical data is needed. We hypothesize that only Argonaute 2 (Ago)-bound miRs function in platelet production and function. Aim 2 will identify and characterize miR-125a-5p and miR-15a-5p mRNA targets in living MKs using an unbiased, transcriptome-wide approach called Ago-HITS-CLIP (high throughput sequencing of cross-linked argonate2 immunoprecipitates). Novel miR-mRNA pairs will be validated in cells and seed regions queried for functional SNPs using public GWAS data sets of cardiovascular disease. Our RNA-seq preliminary data demonstrate IFN-stimulated cul- tured MKs regulates miR and mRNA expression, and we hypothesize that IFN-regulated miRs are associated with IFN responsiveness in MPDs. In Aim 3 we will perform Ago-HITS-CLIP on IFN-treated hematopoietic progenitor and differentiating MKs and test for associations between IFN-inducible miRs and hematologic re- sponse in IFN-treated patients with MPDs. The major impact of this research will be to (1) provide insights into the roles of miR-125a-5p and miR-15a-5p in platelet production and function, (2) characterize active miRs in living normal and IFN-stimulated MKs, and (3) lay groundwork for using miRs to enhance in vitro manufac- ture of platelets, as potential therapeutic targets for thrombocytopenia and thrombocytosis, and for mechanistic studies on pathogenic miRs in MPDs.

摘要/摘要目前关于巨核细胞 (MK) 基因表达的知识主要集中在转录、而在健康状态下，人们对翻译过程的了解相对较少，甚至知之甚少。在疾病状态下，失调的 microRNA (miR) 在炎症和发病机制中发挥着关键作用。几种人类骨髓疾病以及小鼠的过度表达和敲除研究已经鉴定出 miR 然而，miR 在 hu 中 MK 基因表达中的作用。人类健康或疾病仍然知之甚少，这项研究将使用体内、体外、生化和生物研究。研究 miR 对正常和应激 (IFNα) 诱导的 MK 基因表达调节的信息方法。根据我们的初步数据，我们在 154 名健康受试者中发现了与血小板计数和功能密切相关的 miR 敲低和过度表达研究表明 miR-125a-5p 调节前血小板形成 (PPF) miR-15a-5p 调节培养的 MK 中 GPVI 诱导的 MK 整合素激活，并且这两个 miR 都是预- 我们努力控制这些 miR 调节在原代人骨髓 MK 中表达的靶基因。后期体内巨核细胞生成 (MKpoiesis) 和血小板生成和功能将测试这一点。使用 miR-125a 和 miR-15a 的 MK 特异性消融进行体内假设，以及胃肠外给药抗 miR，使用血小板生成、Mkpoiesis 和 GPVI 介导的血小板反应性、血栓的测定要了解 miR 的功能，了解其 mRNA 靶点至关重要。最近，识别 miR 靶点的方法一直基于具有高错误率的计算机算法。阳性和假阴性预测率，因此需要额外的生化数据。只有 Argonaute 2 (Ago) 结合的 miR 在血小板生成和功能中发挥作用，Aim 2 才能识别和发挥作用。使用无偏见的全转录组表征活体 MK 中的 miR-125a-5p 和 miR-15a-5p mRNA 靶标称为 Ago-HITS-CLIP（交联 argonate2 免疫沉淀物高通量测序）的方法。新的 miR-mRNA 对将在使用公共查询功能性 SNP 的细胞和种子区域中进行验证心血管疾病的 GWAS 数据集。我们的 RNA-seq 初步数据证明了 IFNα 刺激的培养物。 turd MKs 调节 miR 和 mRNA 表达，我们已经发现 IFNα 调节的 miRs 与在目标 3 中，我们将对 IFNα 治疗的造血系统进行 Ago-HITS-CLIP。祖细胞和分化 MK 并测试 IFNα 诱导的 miR 与血液学复发之间的关联接受 IFNα 治疗的 MPD 患者的反应这项研究的主要影响将是 (1) 提供见解。研究 miR-125a-5p 和 miR-15a-5p 在血小板生成和功能中的作用，(2) 表征活性 miR (3) 为使用 miR 增强体外生产奠定基础血小板的性质，作为血小板减少症和血小板增多症以及机械性血小板减少症的潜在治疗靶点 MPD 中致病性 miR 的研究。