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

项目负责人：Alain Charest。细胞外RNA（exRNA）是一种新发现的细胞通讯形式， 一个细胞到另一个细胞是通过RNA分子传递的。exRNA被保护免受细胞外RNA酶的侵害， 包封在膜囊泡内或作为RNA和蛋白质复合物的混合物 （核糖核蛋白RNP）。这些实体的产生似乎不是随机的，而是 一个高度协调的机制，其细节仍然不明确。在这份提案中，我们的目标是揭示 exRNA被包裹在细胞外囊泡和RNP中的分子机制以及如何 使用多形性胶质母细胞瘤（GBM）作为模型系统，治疗干预影响这些机制。 使用GBM的基因工程小鼠模型，该模型由GBM的过表达和激活驱动， EGFR和PDGFRa是GBM中发现的两种最常见的遗传事件，我们将确定囊泡和 使用深度测序方法的EGFR GBM和PDGFRa GBM的RNP exRNA谱。一旦 建立后，我们将研究治疗性治疗对exRNA产生动力学的影响， 在体外和体内进行exRNA对靶细胞的功能研究。我们将首先关注miRNA，因为它 已经表明，miRNA构成exRNA的显著比例。 我们的项目在多个层面上与U19应用程序中的其他项目相关。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的exRNA序列组成， 我们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， 胶质母细胞瘤中exRNA产生的分子机制作为EGFR和PDGFRa信号传导的功能 途径，并与Xandra Breakefield博士合作开发研究exRNA的方法和试剂 转移到受体细胞。最后，我们将与Thorsten Mempel博士密切合作，他将提供活体内 成像技术，以评估和研究exRNA荧光报告基因在体内。

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.