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Visualization of exosome-mediated miR-210 from hypoxic tumor cells
저산소 암세포 유래 miR-210의 엑소좀을 통한 전달 영상화

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Authors
정경오
Advisor
정준기
Major
의과대학 의과학과
Issue Date
2016-08
Publisher
서울대학교 대학원
Keywords
Breast cancerexosomehypoxiamiR-210tumor microenvironment
Description
학위논문 (박사)-- 서울대학교 대학원 : 의과학과, 2016. 8. 정준기.
Abstract
Introduction: Cells release exosomes, which are known to carry specific cellular components such as proteins, mRNA, and miRNA for communicating with other cells. Particularly, cancer cells actively communicate with neighboring cancer cells and other cells in the tumor microenvironment. In this study, I constructed a DNA reporter gene vector to visualize miR-210 function, and established cell lines containing this construct. I transferred exosomes from hypoxic cancer cells into breast cancer cells and endothelial cells, and demonstrated the expression and function of miR-210 in the recipient cells.
Methods: To evaluate miR-210 function, I developed a miR-210 specific reporter (pCMV-luc2/miR-210), which was designed such that the luciferase signal was turned off on binding to miR-210. Using this vector, 4T1 (mouse breast cancer cells) and SVEC (mouse endothelial cells) were transfected. Hypoxia was induced by Desferoximine (DFO). Hypoxic exosomes were isolated by ultracentrifugation or ExoQuick, and characterized by western blot analysis and transmission electron microscopy (TEM). Real-time PCR was performed to measure the amount of miR-210. Luciferase activity was measured by luciferase activity assay and IVIS imaging. In tumor tissues, immunohistochemistry was performed for detecting HIF-1a, luciferase, Ephrin-A3, PTP1B, and VEGF.
Results: The amount of miR-210 increased in hypoxic 4T1 cells, and in the exosomes from the hypoxic tumor cells. In bioluminescence imaging, luciferase signals of Exo (+) 4T1 and Exo (+) SVEC cells decreased, as compared to those of Exo (-) cells. In xenograft mouse models, the luciferase signals also decreased from tumors treated with hypoxic exosomes, indicating exosome-mediated transfer of miR-210. The expression of Ephrin-A3 and PTP1B, miR-210 target proteins also decreased in the hypoxic exosome-treated tumor cells, while the expression of VEGF increased.
Conclusion: Transfer of miR-210 through exosomes was successfully visualized from hypoxic cancer cells to cancer cells and endothelial cells, and had it had effects on angiogenesis-related genes. This imaging system can be also applied to understand basic mechanism of intercellular communication between tumor microenvironment cells via exosomes.
Language
English
URI
https://hdl.handle.net/10371/122317
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College of Medicine/School of Medicine (의과대학/대학원)Dept. of Biomedical Sciences (대학원 의과학과)Theses (Ph.D. / Sc.D._의과학과)
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