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利用 nCounter 技术进行高通量同时 mRNA 分析表明，细胞外囊泡中含有不同于其亲代前列腺癌细胞的 mRNA 转录本。

High-Throughput Simultaneous mRNA Profiling Using nCounter Technology Demonstrates That Extracellular Vesicles Contain Different mRNA Transcripts Than Their Parental Prostate Cancer Cells.

机构信息

The Brady Urological Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21218, United States.

Department of Urology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200072, China.

出版信息

Anal Chem. 2021 Mar 2;93(8):3717-3725. doi: 10.1021/acs.analchem.0c03185. Epub 2021 Feb 17.

DOI:10.1021/acs.analchem.0c03185
PMID:33596381
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7944479/
Abstract

Extracellular vesicles (EVs) are nano-sized lipid bilayer encapsulated particles with a molecular cargo that appears to play important roles within the human body, such as in cell-to-cell communication. Unraveling the composition of EV cargos remains one of the most fundamental steps toward understanding the role of EVs in intercellular communication and the discovery of new biomarkers. One of the unmet needs in this field is the lack of a robust, sensitive, and multiplexed method for EV mRNA profiling. We established a new protocol using the NanoString low RNA input nCounter assay by which the targeted mRNA transcripts in EVs can be efficiently and specifically amplified and then assayed for 770 mRNAs in one reaction. Prostate cancer cells with epithelial (PC3-Epi) or mesenchymal (PC3-EMT) phenotypes and their progeny EVs were analyzed by the same panel. Among these mRNAs, 157 were detected in PC3-Epi EVs and 564 were detected in PC3-EMT EVs. NOTCH1 was the most significantly abundant mRNA transcripts in PC3-EMT EVs compared to PC3-Epi EVs. Our results demonstrated that when cells undergo epithelial-to-mesenchymal transition (EMT), a more active loading of cancer progression-related mRNA transcripts may occur. The mRNA cargos of EVs derived from mesenchymal prostate cancer cells may contribute to the pro-EMT function. We found that mRNA transcripts are different in progeny EVs compared to parental cells. EV cargos are not completely reflective of their cell origin, and the underlying mechanism of cargo sorting is complicated and needs to be further elucidated.

摘要

细胞外囊泡 (EVs) 是纳米大小的脂质双层包裹的颗粒，其分子货物似乎在人体内发挥着重要作用，例如在细胞间通讯中。揭示 EV 货物的组成仍然是理解 EV 在细胞间通讯中的作用和发现新的生物标志物的最基本步骤之一。该领域未满足的需求之一是缺乏一种强大、敏感和多重的 EV mRNA 分析方法。我们建立了一种新的使用 NanoString 低 RNA 输入 nCounter 检测的方案，通过该方案，可以有效地和特异性地扩增 EV 中的靶向 mRNA 转录本，然后在一个反应中检测 770 个 mRNA。用相同的试剂盒分析具有上皮 (PC3-Epi) 或间充质 (PC3-EMT) 表型的前列腺癌细胞及其后代 EV。在这些 mRNA 中，在 PC3-Epi EV 中检测到 157 个，在 PC3-EMT EV 中检测到 564 个。与 PC3-Epi EV 相比，NOTCH1 是 PC3-EMT EV 中最丰富的 mRNA 转录本。我们的结果表明，当细胞经历上皮-间充质转化 (EMT) 时，可能会发生更多与癌症进展相关的 mRNA 转录本的主动加载。源自间充质前列腺癌细胞的 EV 的 mRNA 货物可能有助于 EMT 功能。我们发现，与亲本细胞相比，后代 EV 中的 mRNA 转录本不同。EV 货物不完全反映其细胞来源，货物分选的潜在机制很复杂，需要进一步阐明。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ebe4/7944479/82f19cd23e3d/ac0c03185_0007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ebe4/7944479/ffd04b6d7a5d/ac0c03185_0002.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ebe4/7944479/82f19cd23e3d/ac0c03185_0007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ebe4/7944479/ffd04b6d7a5d/ac0c03185_0002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ebe4/7944479/d25ffb8bcd6c/ac0c03185_0003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ebe4/7944479/dc74bdb2af7c/ac0c03185_0004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ebe4/7944479/a15b0d4d88da/ac0c03185_0005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ebe4/7944479/73df4fbc6426/ac0c03185_0006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ebe4/7944479/82f19cd23e3d/ac0c03185_0007.jpg

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