Abstract
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Objectives In this study, we visualized exosome-mediated transfer of hypoxia-induced miR-210 in vitro and in vivo.
Methods Mouse breast cancer cells, 4T1, were transfected with pCMV-luc2/miR-210 which was designed to be turned off luciferase signals by binding of miR-210. Hypoxia was induced by Deferoxamine (DFO). Exosomes were isolated by ultracentrifugation and ExoQuick, and characterized by western blot and TEM. Real-time PCR was performed to measure the amount of miR-210. Luciferase activity was measured by luciferase assay and IVIS imaging. In tissues, immunohistochemistry was performed for HIF-1a, luciferase, Ephrin-A3 and VEGF.
Results In real-time PCR, miR-210 was increased in hypoxic cells (15.70 fold) and exosomes from the media of hypoxic tumor cells (12.73 fold). In xenograft mouse models, luciferase signals from the tumors treated with DFO or hypoxic exosomes were decreased in 0.53 or 0.56 fold compared to control tumor. The expression of Ephrin-A3, a miR-210 target protein, was also decreased in the hypoxic exosome-treated tumor cells, whilst the expression of VEGF was increased in those cells.
Conclusions We developed a reporter gene imaging system for monitoring miR-210. Transfer of miR-210 through exosomes was successfully visualized in vitro as well as in vivo. This imaging system can be useful for monitoring exosomal miRNA transfer in tumor cells.