MicroRNA function in human megakaryocytes

描述(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是病理性血栓形成的原因。血小板基因和蛋白的表达--因此，血小板的产生和反应--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针对mRNAs降解或抑制的microRNAs(MiRNAs)的调控。MiRNAs在所有人类生理学中都是重要的，尽管失调的miRNA生物学导致了血液疾病，但对miRNA在MEGs中的作用知之甚少。我们测量了154名健康受试者的血小板表型、miRNA谱和mRNA谱。这一数据中的表型变异使我们能够定义少数通过GPVI、PAR1、PAR4和P2Y12来调节血小板反应性的特定“主要miRNAs”。我们假设MEG miRNAs调节控制血小板反应性和血小板数量的mRNAs。本研究的总体目标是了解miRNAs在人类Megs(HMegs)中的功能。目标1将描述候选“主”miRNAs调节血小板聚集的功能。候选miRNAs将被转化为从CD34+干细胞分化的hMegs，并测试激动剂诱导的细胞激活。预测的信使核糖核酸结合位点将在MEGS中得到验证，并测试新的信使核糖核酸靶点的功能。MiRNAs是已知的其他细胞中凋亡基因的调节者，但miRNAs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。在更多的初步数据中，我们已经鉴定了与血小板数量相关的特定miRNAs，并敲除了编码也与血小板数量相关的凋亡调节基因的mRNAs(包括BCL2L2[Bcl-w]、MCL1、CASP1等)。目的2将评估miRNAs在MEG细胞凋亡和血小板生成中的作用。候选miRNAs将在hMegs中进行测试，以检测原血小板形成、血小板生成、Meg凋亡和Meg线粒体功能。更多的初步数据表明，某些miRNAs的血细胞类型偏好表达可以决定细胞偏好转基因表达。目的3将评估miRNAs的造血祖细胞优先表达是否有助于MEG优先基因的表达。MiRNA水平将通过激光捕获显微解剖原始人类骨髓细胞以及同一受试者的外周血细胞谱系来描述。细胞偏好miRNAs的结合位点将被工程化为慢病毒载体，该载体将感染造血干细胞，并评估转基因在MEGS和其他谱系中的表达。这些实验将阐明miRNAs在MEG/血小板功能和血小板生成中的作用，加深我们对谱系分化和血小板寿命的分子机制的了解，并为miRNAs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性的方式改变MEG基因的表达奠定基础。

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.