MicroRNA function in human megakaryocytes

描述（由申请方提供）：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--很大程度上受到巨核细胞（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由靶向mRNA进行降解或抑制的microRNA（miRNAs）调节。miRNA在所有人类生理学中都是重要的，尽管失调的miRNA生物学有助于血液疾病，但对Megs中的miRNA作用知之甚少。我们测量了154名健康受试者的血小板表型、miRNA谱和mRNA谱。该数据中的表型变异允许我们定义少量通过GPVI、PAR 1、PAR 4和P2 Y12调节血小板反应性的特异性“主miRNA”。我们假设Meg miRNAs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miRNAs在人类Megs（hMegs）中的功能。目的1将表征候选“主”miRNAs调节血小板聚集的功能。将候选miRNA转导至从CD 34+干细胞分化的hMegs中，并测试激动剂诱导的细胞活化。将验证预测的mRNA结合位点，并在Megs中测试新型mRNA靶标的功能。miRNAs是其他细胞中细胞凋亡基因的既定调节因子，但miRNAs在调节hMeg细胞凋亡或血小板减少调节的血小板生成中没有已知的作用。在额外的初步数据中，我们已经鉴定了与血小板数量相关的特定miRNA，其靶向并敲除编码与血小板数量也相关的血小板减少调节基因（包括BCL 2L 2 [Bcl-w]，MCL 1，CASP 1等）的mRNA。目的2探讨miRNAs在Meg细胞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、血小板产生、Meg凋亡和Meg线粒体功能。额外的初步数据表明，血细胞类型优先表达的一些miRNA可以决定细胞优先转基因表达。目的3将评估造血祖细胞优先表达的miRNA是否有助于Meg-preferential基因表达。将通过激光捕获显微切割原代人骨髓细胞以及来自同一受试者的外周血细胞谱系来分析miRNA水平。将细胞优先miRNA的结合位点工程化到慢病毒载体中，其将感染造血干细胞，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作为生物标志物和潜在的治疗工具在血栓形成和止血疾病中以谱系限制的方式改变Meg基因表达奠定基础。

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.