Mouse models of glioma to study functional exRNA transfer to the microenvironmen

项目负责人：阿兰·查雷斯特。细胞外RNA(ExRNA)是一种新发现的细胞通信形式，通过这种形式，来自 一个细胞到另一个细胞的运输是由RNA分子完成的。外源RNA通过以下方式免受细胞外核糖核酸酶的影响 囊化在膜囊泡内或作为RNA和蛋白质复合体的融合 (核糖核蛋白RNPs)。这些实体的产生似乎不是随机的，是以下因素的结果 这是一台高度协调的机器，其细节仍未明确。在这项提案中，我们的目标是揭示 外源RNA被包裹在胞外小泡和RNPs中的分子机制及其机制 以多形性胶质母细胞瘤(GBM)为模型系统，治疗干预措施影响这些机制。 使用基因工程小鼠的GBM模型，该模型由过度表达和激活的 EGFR和PDGFRA，这两个在GBM中发现的最常见的遗传事件，我们将确定囊泡和 利用深度测序方法对EGFR基底膜和PDGFRA基底膜的RNP exRNA图谱进行分析。一次 ，然后我们将研究治疗对exRNA产生和动力学的影响 在体外和体内对靶细胞进行外源RNA的功能研究。我们将首先关注miRNA，因为它 已有研究表明，miRNA在exRNA中占有很大比例。 我们的项目与这个U19申请中的其他项目在多个层面上相关。我们将密切合作 与安娜·克里切夫斯基博士合作破译和编目外源RNA序列组成 我们的基因定义的胶质母细胞瘤肿瘤细胞。我们还将与斯蒂芬·古尔德博士合作 EGFR和PDGFRA信号在胶质母细胞瘤中产生exRNA的分子机制 与Xandra Breakefield博士共同开发研究exRNA的方法和试剂 转移到受体细胞。最后，我们将与托尔斯滕·门佩尔博士密切合作，他将在生命周期内提供 成像技术在体内评价和研究外源RNA荧光报告。

Project Leader: Alain Charest. Extracellular RNA (exRNA) is a newly discovered form of cellular communication whereby information from one cell to another is conveyed by RNA molecules. exRNAs are protected from extracellular RNases by encapsulation within membrane vesicles or as amalgamations of RNA and proteins complexes (ribonucleoproteins RNPs). It appears that the production of these entities is not random and is the result of a highly orchestrated machinery, the details of which remain ill-defined. In this proposal, we aim to uncover the molecular mechanisms by which exRNA is encapsulated in extracellular vesicles and RNPs and how therapeutic interventions affect these mechanisms using glioblastoma multiforme (GBM) as a model system. Using genetically engineered mouse models of GBM that are driven by overexpression and activation of EGFR and PDGFRa, the two most common.genetic events found in GBM, we will determine the vesicle and RNP exRNA profiles of EGFR GBMs and PDGFRa GBMs using deep sequencing methods. Once established, we will then study the effect of therapeutic treatment on the dynamics of exRNA production and perform functional studies of exRNA on target cells in vitro and in vivo. We will initially focus on miRNA as it has been shown that miRNA make up a significant proportion of exRNA. Our project relates to the other projects in this U19 application on multiple levels. We will work in close collaboration with Dr. Anna Krichevsky on deciphering and cataloging the exRNA sequence composition of our genetically-defined glioblastoma tumor cells. We will also collaborate with Dr. Stephen Gould on the molecular mechanisms of exRNA production in glioblastoma as a function of EGFR and PDGFRa signaling pathways and with Dr. Xandra Breakefield on the development of methods and reagents to study exRNA transfer to recipient cells. Finally, we will work closely with Dr. Thorsten Mempel who will provide intravital imaging technology to evaluate and study exRNA fluorescent reporters in vivo.