エクソソームを介したトランスポゾン活性の発がんへの影響

外泌体介导的转座子活性对致癌作用的影响

基本信息

批准号：
18J11869
负责人：
河村有美
金额：
$ 0.96万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for JSPS Fellows
财政年份：
2018
资助国家：
日本
起止时间：
2018-04-25 至 2020-03-31
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18J11869/
关键词：
extracellular vesicle retrotransposon horizontal gene transfer

项目摘要

Long interspersed element-1 (LINE-1 or L1) retrotransposons replicate through a copy-and-paste mechanism using an RNA intermediate. However, little is known about the physical transmission of retrotransposon RNA between cells. To examine the horizontal transfer of an active human L1 retrotransposon mediated by extracellular vesicles (EVs), human cancer cells were transfected with an expression construct containing a retrotransposition-competent human L1 tagged with a reporter gene. Using this model, active retrotransposition events were detected by screening for the expression of the reporter gene inserted into the host genome by retrotransposition. EVs including exosomes and microvesicles were isolated from cells by differential centrifugation. The enrichment of L1-derived reporter RNA transcripts were detected in EVs isolated from cells expressing active L1 retrotransposition. The delivery of reporter RNA was confirmed in recipient cells, and reporter genes were detected in the genome of recipient cells. Additionally, employing qRT-PCR, we found that host-encoded factors are activated in response to increased exposure to L1-derived RNA transcripts in recipient cells. Our results suggest that the horizontal transfer of retrotransposons can occur through the incorporation of RNA intermediates delivered via EVs and may have important implications for the intercellular regulation of gene expression and gene function.

长散布的 element-1（LINE-1 或 L1）反转录转座子通过使用 RNA 中间体的复制粘贴机制进行复制。然而，人们对逆转录转座子 RNA 在细胞之间的物理传递知之甚少。为了检查由细胞外囊泡 (EV) 介导的活性人 L1 逆转录转座子的水平转移，用含有报告基因标记的具有逆转录转座能力的人 L1 的表达构建体转染人癌细胞。使用该模型，通过筛选通过逆转录转座插入宿主基因组的报告基因的表达来检测主动逆转录事件。通过差速离心从细胞中分离出包括外泌体和微泡的 EV。在从表达活性 L1 逆转录转座的细胞中分离的 EV 中检测到 L1 衍生的报告 RNA 转录本的富集。报告RNA的递送在受体细胞中得到证实，并且在受体细胞的基因组中检测到报告基因。此外，利用 qRT-PCR，我们发现宿主编码的因子会随着受体细胞中 L1 衍生 RNA 转录物的暴露增加而被激活。我们的结果表明，逆转录转座子的水平转移可以通过 EV 传递的 RNA 中间体的掺入来发生，并且可能对基因表达和基因功能的细胞间调节具有重要意义。