Functional changes in secreted RNA biogenesis using in vivo colon tumor models

这一提议的一个主要前提是，存在于生物体液中的RNA代表着进入各种细胞的窗口 存在于多种组织中的释放这类RNA的过程。我们发现致癌的KRAS 发生在结直肠癌中的突变可以调节肿瘤分泌的miRNAs、mRNAs和Long RNAs 外体中的细胞。本项目(项目1)将确定这些RNAs通过什么机制 (CRNAs)使用体内模型进入血流，以及这些RNA如何随着致癌突变而变化。 该项目将导致创建新的工具，以实时和从 小鼠模型的血液样本。为了解决这些问题，我们提出了以下建议。 目的1.测定正常人和结直肠癌患者血浆中循环RNA的含量。 我们假设，与正常细胞相比，大肠肿瘤细胞分泌的循环RNA(CRNAs)成分发生了变化。此外，这些变化中的许多将从小鼠保存到人类CRC。这一目标的目的是识别随着肿瘤进展(晚期腺瘤和癌症)而改变的血浆CRNAs。 目的2.阐明肿瘤RNA进入循环的途径(S)。 不同的机制可能参与了CRC诱导的cRNA改变。在这个目标中，我们将确定目标1中确定的外切体相关的miRNAs和长RNAs进入循环的途径。 目的3.研究肿瘤基因对人原发肿瘤移植瘤CRNAs的影响，以及候选运输蛋白和相互作用基序在改变肿瘤RNA分泌中的作用。 结合在项目2中进行的研究，我们将确定RNA被贩运到外体并分泌到血液中的机制。在这个目标中，我们将确定人类结直肠癌异种移植的基因如何影响外体相关的CRNAs。

A major premise of this proposal is that RNAs present in biofluids represent a window into various cellular processes that exist in multiple tissues that release such RNAs. We have found that oncogenic KRAS mutations that occur in colorectal cancer can regulate miRNAs, mRNAs and long RNAs secreted from tumor cells in exosomes. This project (project 1) will determine what the mechanisms are by which these RNAs (cRNAs) enter the bloodstream using in vivo models and how these RNAs change with oncogenic mutations. This project will result in the creation of new tools to visualize and analyze such RNAs in real time and from blood samples in mouse models. We propose the following aims to address these questions. Aim 1. Determine circulating RNAs in human and mouse plasma that differ in normal individuals and those bearing colorectal tumors. We hypothesize that colorectal tumor cells secrete an altered composition of circulating RNAs (cRNAs) compared to normal cells. Further, many of these changes will be conserved from mouse to human CRC. The goal of this aim is to identify plasma cRNAs that are altered with tumor progression (advanced adenomas and carcinomas). Aim 2. Elucidate the pathway(s) by which tumor RNAs traffic into the circulation. Various mechanisms may contribute to CRC-induced cRNA alterations. In this aim, we will determine the route by which the exosome-associated miRNAs and long RNAs identified in Aim 1 enter the circulation. Aim 3. Determine the influence of tumor genotype on cRNAs using human primary tumor xenografts and determine the function of candidate trafficking proteins and interacting motifs in altering tumor RNA secretion. In conjunction with studies performed in Project 2, we will determine the mechanism by which RNAs are trafficked into exosomes and secreted into the blood. In this aim, we will determine how the genotype of human CRC xenografts affects exosome-associated cRNAs.