MicroRNA function in human megakaryocytes

MicroRNA 在人类巨核细胞中的功能

• 批准号: 8984318
• 负责人: PAUL F. BRAY
• 金额: $ 45.31万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2016-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8984318
• 关键词: 3' Untranslated Regions; Agonist; Apoptosis; Apoptosis Regulation Gene; Binding Sites; Biological; Biology; Blood Cells; Blood Platelets; Bone Marrow Cells; CASP1 gene; CASP8 gene; CD34 gene; Cell Lineage; Cells; Cellular biology; Code; Conflict (Psychology); Data; Data Set; Disease; Dissection; Engineering; Environment; F2R gene; Future; Gene Expression; Genes; Genetic Transcription; Goals; Health; Hematological Disease; Hematopoietic; Hematopoietic stem cells; Hemorrhage; Hemostatic function; Human; Integrins; Knowledge; Lasers; Lentivirus Vector; Longevity; MCL1 gene; Measures; Megakaryocytes; Messenger RNA; MicroRNAs; Mitochondria; Molecular; Myocardial Infarction; PAWR gene; Pathologic; Phenotype; Physiology; Plasma; Platelet Count measurement; Platelet aggregation; Production; Proteins; RNA; Reporter; Reporter Genes; Research; Role; Staging; Stem cells; Stroke; System; Testing; Therapeutic; Thrombopoiesis; Thrombosis; Transgenes; Translation Process; Variant; base; cell type; clinically relevant; design; gene function; human data; improved; induced pluripotent stem cell; insight; interest; knock-down; mRNA Transcript Degradation; microvesicles; novel; novel strategies; potential biomarker; progenitor; protein expression; research study; targeted treatment; tool; transgene expression

DESCRIPTION (provided by applicant): Reduced platelet numbers and function are common causes of pathologic bleeding, whereas increased platelet numbers and reactivity are believed to contribute to pathologic thrombosis. Platelet gene and protein expression - and hence, platelet production and reactivity - are largely controlled by megakaryocyte (Meg) transcription, post-transcriptional processing and translation. The expression of most protein-coding genes is regulated by microRNAs (miRNAs) that target mRNAs for degradation or inhibition. MiRNAs are important in all human physiology, and although dysregulated miRNA biology contributes to hematologic diseases, little is known about miRNA effects in Megs. We have measured platelet phenotypes, miRNA profiles and mRNA profiles on 154 healthy subjects. The phenotypic variation in this data allowed us to define a small number of specific "master miRNAs" regulating platelet reactivity through GPVI, PAR1, PAR4 and P2Y12. We hypothesize that Meg miRNAs regulate mRNAs that control platelet reactivity and platelet number. The overall goal of this research is to understand the function of miRNAs in human Megs (hMegs). Aim 1 will characterize the functionality of candidate "master" miRNAs regulating platelet aggregation. Candidate miRNAs will be transduced into hMegs differentiated from CD34+ stem cells and tested for agonist-induced cell activation. Predicted mRNA binding sites will be validated and function of novel mRNA targets tested in Megs. MiRNAs are established regulators of apoptosis genes in other cells, but miRNAs have no known role in regulating hMeg apoptosis or apoptosis-regulated thrombopoiesis. In additional preliminary data we have identified specific miRNAs associated with platelet number that target and knock down mRNAs encoding apoptosis-regulating genes also associated with platelet number (including BCL2L2 [Bcl-w], MCL1, CASP1 and others). Aim 2 will assess the role of miRNAs in Meg apoptosis and thrombopoiesis. Candidate miRNAs will be tested in hMegs for proplatelet formation, platelet production, Meg apoptosis and Meg mitochondrial function. Additional preliminary data indicates that blood cell type-preferential expression of some miRNAs can dictate cell-preferential transgene expression. Aim 3 will assess whether hematopoietic progenitor-preferential expression of miRNAs contributes to Meg-preferential gene expression. MiRNA levels will be profiled from laser-capture micro-dissection of primary human bone marrow cells, as well as peripheral blood cell lineages from the same subject. Binding sites for cell-preferential miRNAs will be engineered into lentiviral vectors, which will be infect hematopoietic stem cells and transgenes expression assessed in Megs and other lineages. The experiments in this proposal will clarify the role of miRNAs in Meg/platelet function and thrombopoiesis, improve our understanding of molecular mechanisms of lineage differentiation and platelet life span, and lay groundwork for miRNAs as biomarkers and potential therapeutic tools for altering Meg gene expression in a lineage-restricted manner in disorders of thrombosis and hemostasis.

描述（由申请人提供）：血小板数量和功能减少是病理性出血的常见原因，而血小板数量和反应性增加被认为有助于病理性血栓形成。血小板基因和蛋白质表达（以及血小板的产生和反应性）在很大程度上受巨核细胞 (Meg) 转录、转录后加工和翻译的控制。大多数蛋白质编码基因的表达受到 microRNA (miRNA) 的调节，这些 microRNA 以 mRNA 为目标进行降解或抑制。 miRNA 在所有人类生理学中都很重要，尽管 miRNA 生物学失调会导致血液疾病，但人们对 miRNA 对 Meg 的影响知之甚少。我们测量了 154 名健康受试者的血小板表型、miRNA 谱和 mRNA 谱。这些数据中的表型变异使我们能够定义少量通过 GPVI、PAR1、PAR4 和 P2Y12 调节血小板反应性的特定“主 miRNA”。我们假设 Meg miRNA 调节控制血小板反应性和血小板数量的 mRNA。本研究的总体目标是了解 miRNA 在人类 Megs (hMegs) 中的功能。目标 1 将表征候选“主”miRNA 调节血小板聚集的功能。候选 miRNA 将被转导为从 CD34+ 干细胞分化而来的 hMegs，并测试激动剂诱导的细胞激活。预测的 mRNA 结合位点将得到验证，新的 mRNA 靶点的功能将在 Megs 中进行测试。 miRNA 是其他细胞中凋亡基因的既定调节因子，但 miRNA 在调节 hMeg 凋亡或凋亡调节的血小板生成中的作用尚不清楚。在额外的初步数据中，我们鉴定了与血小板数量相关的特定 miRNA，这些 miRNA 靶向并敲低编码也与血小板数量相关的凋亡调节基因的 mRNA（包括 BCL2L2 [Bcl-w]、MCL1、CASP1 等）。目标 2 将评估 miRNA 在 Meg 凋亡和血小板生成中的作用。将在 hMegs 中测试候选 miRNA 的前血小板形成、血小板生成、Meg 凋亡和 Meg 线粒体功能。其他初步数据表明，一些 miRNA 的血细胞类型优先表达可以决定细胞优先转基因表达。目标 3 将评估 miRNA 的造血祖细胞优先表达是否有助于 Meg 优先基因表达。 miRNA 水平将通过激光捕获的原代人骨髓细胞以及来自同一受试者的外周血细胞谱系的显微解剖来分析。细胞优先的 miRNA 的结合位点将被设计成慢病毒载体，该载体将感染造血干细胞并在 Megs 和其他谱系中评估转基因表达。本提案中的实验将阐明 miRNA 在 Meg/血小板功能和血小板生成中的作用，提高我们对谱系分化和血小板寿命的分子机制的理解，并为 miRNA 作为改变 Meg 基因表达的生物标志物和潜在治疗工具奠定基础。血栓形成和止血疾病中的谱系限制方式。