Abstract
MiRNAs in the circulation have been demonstrated to be a type of signaling molecule involved in intercellular communication but little is known about their role in regulating radiosensitivity. This study aims to investigate the effects of extracellular miRNAs induced by ionizing radiation (IR) on cell proliferation and radiosensitivity. The miRNAs in the conditioned medium (CM) from irradiated and non-irradiated A549 lung cancer cells were compared using a microarray assay and the profiles of 21 miRNAs up and down-regulated by radiation were confirmed by qRT-PCR. One of these miRNAs, miR-1246, was especially abundant outside the cells and had a much higher level compared with that inside of cells. The expressions of miR-1246 in both A549 and H446 cells increased along with irradiation dose and the time post-irradiation. By labeling exosomes and miR-1246 with different fluorescence dyes, it was found that the extracellular miR-1246 could shuttle from its donor cells to other recipient cells by a non-exosome associated pathway. Moreover, the treatments of cells with miR-1246 mimic or its antisense inhibitor showed that the extracellular miR-1246 could enhance the proliferation and radioresistance of lung cancer cells. A luciferase reporter-gene transfer experiment demonstrated that the death receptor 5 (DR5) was the direct target of miR-1246, and the kinetics of DR5 expression was opposite to that of miR-1246 in the irradiated cells. Our results show that the oncogene-like extracellular miR-1246 could act as a signaling messenger between irradiated and non-irradiated cells, more importantly, it contributes to cell radioresistance by directly suppressing the DR5 gene.
Highlights
Radiotherapy treatment is widely used for the treatment of malignant tumors, radioresistance remains a major clinical obstacle to a successful cancer therapy and it may lead to poor prognosis of radiotherapy
To determine which extracellular miRNA expressions could be significantly changed in lung cancer cells in response to ionizing radiation (IR), miRNAs in the conditioned medium (CM) collected from irradiated A549 cells at 24 h after 4 Gy irradiation were compared with that from nonirradiated control by using the Agilent Human miRNA microarray containing 2006 human miRNA probes
The expression levels of different miRNAs had some variability in duplicate experiments, a total of 17 upregulated miRNAs and 4 down-regulated miRNAs were detected in the CM of irradiated cells compared with control
Summary
Radiotherapy treatment is widely used for the treatment of malignant tumors, radioresistance remains a major clinical obstacle to a successful cancer therapy and it may lead to poor prognosis of radiotherapy. It has been reported that lung cancer could effectively evade radiation-mediated cell killing by a number of strategies including radioresistance and tumor microenvironment mediated signaling [1, 2]. Association with exosomes is the dominant model of extracellular miRNA stably existing in different bio-fluids [7,8,9,10]. A significant portion of extracellular miRNAs stay in non-exosome associated structures including the complexes combined with argonaute 2 (AGO2) [11], nucleophosmin-1 (NPM1) [12] and high www.impactjournals.com/oncotarget density lipoprotein (HDL) [13]. The findings of stable extracellular miRNAs in body fluid raises great interest in the physiological function of such external miRNAs
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