Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs.
Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs.
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DOI：
10.1371/journal.pone.0259563
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Ding WQ
中科院分区：
文献类型：
--
作者：
Xu YF;Xu X;Bhandari K;Gin A;Rao CV;Morris KT;Hannafon BN;Ding WQ
细胞外囊泡（ev）中的microRNAs （miRNA）已被研究作为胰腺导管腺癌（PDAC）的潜在生物标志物。然而，通常使用常规的外泌体分离方法获得混合的ev群体用于生物标志物的开发。电动汽车来源于不同的细胞过程，存在不同的大小，因此它们之间的miRNA表达无疑是不同的。我们开发了一种简单的方案，利用顺序过滤和超离心将PDAC ev分为三组，一组平均直径大于220 nm，命名为操作3 (OP3)；平均直径在100-220纳米之间，称为OP2 (operational 2)；另一种平均直径约为100纳米，称为OP1 （operational 1）。从人PDAC异种移植小鼠和早期PDAC患者的条件细胞培养基和血浆中分离出ev，并通过纳米颗粒跟踪、western blot和电子显微镜进行验证。我们证明外泌体特异性标记仅在OP1组中富集。来自PDAC细胞的EV中miRNA表达的qRT-PCR分析显示，与其他EV组相比，OP1组中miR-196a和miR-1246的表达显著升高，这两种先前鉴定的miRNA在PDAC细胞来源的外泌体中高度富集。用PDAC异种移植小鼠和局部PDAC患者的血浆ev证实了这一点。我们的研究结果表明，OP1可以用于识别循环EV miRNA特征，作为PDAC的潜在生物标志物。
microRNAs (miRNA) in extracellular vesicles (EVs) have been investigated as potential biomarkers for pancreatic ductal adenocarcinoma (PDAC). However, a mixed population of EVs is often obtained using conventional exosome isolation methods for biomarker development. EVs are derived from different cellular processes and present in various sizes, therefore miRNA expression among them is undoubtedly different. We developed a simple protocol utilizing sequential filtration and ultracentrifugation to separate PDAC EVs into three groups, one with an average diameter of more than 220 nm, named operational 3 (OP3); one with average diameters between 100–220 nm, named operational 2 (OP2); and another with average diameters around 100 nm, named operational 1 (OP1)). EVs were isolated from conditioned cell culture media and plasma of human PDAC xenograft mice and early stage PDAC patients, and verified by nanoparticle tracking, western blot, and electronic microscopy. We demonstrate that exosome specific markers are only enriched in the OP1 group. qRT-PCR analysis of miRNA expression in EVs from PDAC cells revealed that expression of miR-196a and miR-1246, two previously identified miRNAs highly enriched in PDAC cell-derived exosomes, is significantly elevated in the OP1 group relative to the other EV groups. This was confirmed using plasma EVs from PDAC xenograft mice and patients with localized PDAC. Our results indicate that OP1 can be utilized for the identification of circulating EV miRNA signatures as potential biomarkers for PDAC.
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影响因子：
9.7
作者：
Lai X;Wang M;McElyea SD;Sherman S;House M;Korc M
通讯作者：
Korc M
影响因子：
--
作者：
Chen, Christopher Y.;Hogan, Marie C.;Ward, Christopher J.
通讯作者：
Ward, Christopher J.
DOI：
10.1002/wrna.1413
发表时间：
2017-07
期刊：
Wiley interdisciplinary reviews. RNA
影响因子：
--
作者：
Kim KM;Abdelmohsen K;Mustapic M;Kapogiannis D;Gorospe M
通讯作者：
Gorospe M
DOI：
10.1038/nri3622
发表时间：
2014-03
期刊：
Nature reviews. Immunology
影响因子：
--
作者：
通讯作者：
--
DOI：
10.1158/1940-6207.capr-15-0094
发表时间：
2015-09
期刊：
Cancer prevention research (Philadelphia, Pa.)
影响因子：
--
作者：
Permuth-Wey J;Chen DT;Fulp WJ;Yoder SJ;Zhang Y;Georgeades C;Husain K;Centeno BA;Magliocco AM;Coppola D;Malafa M
通讯作者：
Malafa M