Screening of exosomal microRNAs from colorectal cancer cells
Screening of exosomal microRNAs from colorectal cancer cells
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DOI：
10.3233/cbm-160659
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发表时间：
2016-01-01
影响因子：
3.1
通讯作者：
Dwyer, Roisin M.
中科院分区：
文献类型：
--
作者：
Clancy, Cillian;Khan, Sonja;Dwyer, Roisin M.
背景：细胞释放细胞外膜囊泡，包括称为外小体的微囊泡。目的：鉴定2株结直肠癌细胞分泌的包裹外切体的全序列miRNAs，并研究高表达miRNAs的外切体的工程设计。方法：从HCT-116和HT-29细胞中分离得到外切体。从外切体中提取RNA，制作microRNA阵列。培养细胞表达miR-379(HCT-116-379)或非靶向对照(HCT-116-NTC)，并测定其功能效应。结果：观察到细胞株分泌的40-100 nm的微泡，证实表达外切体蛋白CD63。HT-29外切体含有409个miRNAs，HCT-116外切体含有393个miRNAs，338个外切体是两个细胞系的共同外切体。对选定的目标进行了验证。与对照组相比，Hct-116379细胞表现出增殖减少(减少12-15%，p<；0.001)和迁移减少(32-86%，p<；0.001)。HCT-116-379外切体富含miR-379。共聚焦显微镜观察到HCT-116-379外切体转移到受体细胞。结论：结直肠癌细胞分泌大量miRNAs。MiR-379可以减少细胞的增殖和迁移，并且可以设计出富含miR-379的外切体。
BACKGROUND: Cells release extracellular membrane vesicles including microvesicles known as exosomes. Exosomes contain microRNAs (miRNAs) however the full range within colorectal cancer cell secreted exosomes is unknown.OBJECTIVE: To identify the full range of exosome encapsulated miRNAs secreted from 2 colorectal cancer cell lines and to investigate engineering of exosomes over-expressing miRNAs.METHODS: Exosomes were isolated from HCT-116 and HT-29 cell lines. RNA was extracted from exosomes and microRNA array performed. Cells were engineered to express miR-379 (HCT-116-379) or a non-targeting control (HCT-116-NTC) and functional effects were determined. Exosomes secreted by engineered cells were transferred to recipient cells and the impact examined.RESULTS: Microvesicles 40-100 nm in size secreted by cell lines were visualised and confirmed to express exosomal protein CD63. HT-29 exosomes contained 409 miRNAs, HCT-116 exosomes contained 393, and 338 were common to exosomes from both cell lines. Selected targets were validated. HCT-116-379 cells showed decreased proliferation (12-15% decrease, p < 0.001) and decreased migration (32-86% decrease, p < 0.001) compared to controls. HCT-116-379 exosomes were enriched for miR-379. Confocal microscopy visualised transfer of HCT-116-379 exosomes to recipient cells.CONCLUSIONS: Colorectal cancer cells secrete a large number of miRNAs within exosomes. miR-379 decreases cell proliferation and migration, and miR-379 enriched exosomes can be engineered.