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

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上， 而在健康状态下，翻译过程的了解相对较少，甚至更少 在疾病状态下。失调的微小RNA（miRs）在炎症和炎症的发病机制中起关键作用。 几种人类骨髓疾病，以及在小鼠中的过表达和敲除研究已经鉴定了miR 导致骨髓增生性疾病（MPD）。尽管如此，miR在人巨噬细胞MK基因表达中的作用仍有待进一步研究。 对人类健康或疾病的了解仍然很少。这项研究将使用体内，体外，生化和生物- 研究正常和应激（IFN γ）诱导的MK基因表达的miR调节的信息学方法。在 根据我们的初步数据，我们在154只健康犬中发现了与血小板计数和功能密切相关的miR， nors。敲低和过表达研究显示miR-125 a-5 p调节前血小板形成（PPF） 而miR-15 a-5 p调节培养的MK中GPVI诱导的MK整合素活化，并且两种miR都是前整合素。 指示靶向在原代人骨髓MK中表达的基因。我们假设这些miR调节- 晚期体内巨核细胞生成（MK生成）和血小板产生和功能。目标1将测试此hy- 使用miR-125 a和miR-15 a的MK-特异性消融以及miR-125 a和miR-15 a的肠胃外施用的体内合成 抗miR，使用血小板生成、Mkpoiesis和GPVI介导的血小板反应性、血栓形成和血小板聚集的测定， 形成和信号。为了了解miR如何发挥作用，了解其mRNA靶点至关重要。直到重新- 目前，用于鉴定miR靶点的方法是基于具有高假阳性率的计算机算法， 阳性和假阴性预测率，因此需要额外的生化数据。我们假设 只有Argonaute 2（Ago）结合的miR在血小板产生和功能中起作用。目标2将确定和 使用无偏的全转录组方法表征活MK中的miR-125 a-5 p和miR-15 a-5 p mRNA靶点 这种方法称为Ago-HITS-CLIP（交联argonate 2免疫沉淀物的高通量测序）。 新的miR-mRNA对将在细胞和种子区域中进行验证，使用公共 GWAS心血管疾病数据集。我们的RNA-seq初步数据表明IFN γ刺激的cul-2表达。 受干扰的MK调节miR和mRNA的表达，我们假设IFN γ调节的miR与 MPDs中IFN γ的反应性。在目标3中，我们将对IFN γ处理的造血细胞进行Ago-HITS-CLIP， 祖细胞和分化的MK，并测试IFN γ诱导的miR和血液学再分化之间的关联。 IFN-γ治疗的MPD患者中的反应。这项研究的主要影响将是（1）提供见解 miR-125 a-5 p和miR-15 a-5 p在血小板生成和功能中的作用，（2）表征活性miR 活的正常和IFN γ刺激的MK，和（3）奠定基础，使用miR，以提高在体外制造， 血小板作为血小板减少症和血小板增多症的潜在治疗靶点， MPD中致病性miR的研究。

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.