Tumor-derived exosomes transport miRNA and mRNA for intercellular communication

• Main Authors: Jung Long Wu, 吳俊龍
• Other Authors: Y. S. Chang
• Format: Others
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/63652024537523288445

碩士 === 長庚大學 === 生物醫學研究所 === 98 === Recent studies show that cells release different types of membrane-bound vesicles into extracellular space to serve as mediators for cell-cell communication. Among these secreted vesicles, exosomes are vesicles of endocytic origin with 30 to 100 nm in diameter and contain proteins and nucleic acids as their cargos. Exosomes were shown to be secreted by various cell types, including immune cells and cancer cells. These small membrane vesicles can participate in biological processes such as antigen presentation, cell proliferation and immune reactions.This study aims to characterize the composition of microRNAs in secreted exosomes and to investigate whether these exosomal microRNAs can function asmediators of intercellular gene regulation. We established the ultracentrifugation method to purify exosomes secreted from cultured HeLa cells. Electron microscopic study was conducted to determine the purity, shape and size of the purified exosomes. Biochemical analyses were carried out to verify their protein compositions. Using the stem-loop RT-PCR assay, we identified approximately 80 mature human microRNAs in the purified exosomes. To test whether the secreted exosomes can be uptaken by their target cells, we label the purified exosomes with a fluorescent dye, PKH67, and incubated the labeled exosomes with cultured cancer cells. Our results showed that labeled purified exosomes can be readily detected inside their target cells. Additionally, we purifed exosomes from HeLa cells over-expressing a viral microRNA ebv-miR-BART18-5p. Using the RT-PCR assay, we demonstrated that ebv-miR-BART18-5p can be detected in the HeLa-derived exosomes and can be transferred into the target cells. Using a luciferase reporter assay, we further demonstrated that the exosomal ebv-miR-BART18-5p, once uptaken by the target cells, can effectively suppress its reporter gene inside the target cells. In summary, we have identified the expression pattern of microRNAs in HeLa-derived exosomes and showed that the exosomal microRNAs can function as effective intercellular gene silencing mediators.