KRAS-regulated Ago2-mediated sorting of extracellular RNAs into exosomes

项目摘要/摘要 大约30-50%的大肠肿瘤携带激活的KRAS错义突变。晚期内生体- 衍生的外切体通常由癌细胞分泌，并作为细胞间通讯的媒介。 据报道，外切体与肿瘤的侵袭和转移有关。我们有 以往报道，结直肠癌细胞KRAS突变状态对细胞外的影响很大 外切体的蛋白质和RNA组成。我们最近还报道了ArgAerte 2(Ago2)和 在结肠癌细胞中，一些与Ago2相关的miRNAs到外切体是由KRAS-MEK-Erk信号控制的。 具体地说，Ago2在丝氨酸387上的磷酸化阻止了Ago2与多囊泡内小体的结合 (Mve)和分选成外体。这一发现确定了一种分子机制，它有可能 控制多种miRNAs、miRNA相关蛋白和潜在的其他类型的RNAs的分泌 变成外体。在这个提议的项目中，我将确定ArgAerte 2在将RNA分类到 Exosome，包括使用这种Ago2-来确定在Exosome中分泌的miRNAs的比例 以及miRNA分选中是否存在选择性。我还会决定是否 其他RNA依赖于Ago2来分类成外切体。最后，我将调查功能后果 Ago2介导的KRAS依赖的结肠癌细胞RNA分泌对受体细胞基因表达的影响 以及体内和体外的表型。 好了！

Project Summary/Abstract Approximately 30-50% of colorectal tumors carry activating missense mutations in KRAS. Late endosome- derived exosomes are often secreted from cancer cells and serve as mediators of cell-cell communication. Exosomes have been reported to be associated with tumor aggressiveness and metastasis. We have previously reported that the mutant KRAS status of colorectal cancer cells greatly affects the extracellular protein and RNA composition of exosomes. We also recently reported that sorting of Argonaute 2 (Ago2) and several Ago2-associated miRNAs to exosomes is controlled by KRAS-MEK-Erk signaling in colon cancer cells. Specifically, phosphorylation of Ago2 on serine 387 prevents Ago2 association with multivesicular endosomes (MVE) and sorting into exosomes. This finding identifies a molecular mechanism that has the potential to control the secretion of multiple miRNAs, miRNA-associated proteins, and potentially additional types of RNAs into exosomes. In this proposed project, I will identify the exact role of Argonaute 2 in sorting RNAs to exosomes, including determining what proportion of miRNAs are secreted in exosomes using this Ago2- controlled mechanism as well as whether there is selectivity in miRNA sorting. I will also determine whether other RNAs rely on Ago2 for their sorting into exosomes. Finally, I will investigate the functional consequence of Ago2-mediated RNA secretion from KRAS-dependent colon cancer cells on recipient cell gene expression and phenotypes both in vitro and in vivo. !