Bystander Effects Mediated By Radiation-induced Exosomal MiRNA In Human Bronchial Epithelial Cells

With the development and application of nuclear technology, more and more radiation events impact on people’s daily lives at everywhere. The radiotherapy is a common of treatment to the malignant tumors, comparing to surgery and chemotherapy for it has its own advantages. It is known that the harmful effects from irradiation not only come from the radiation-targeted cells but also from the bystander effect of radiation.As early as 1992, scientists have reported the radiation induced damage effects not only occur in the directly irradiated the nucleus and cytoplasm, but also happen in unexposed/non-targeted contacted neighboring cells or adjacent cells, named radiation-induced bystander effects. These effects often cause the surrounding normal cells and tissues serious harm and threat to human health. Recent studies have found that the radiation-induced some cytokines and reactive oxygen species or other small molecules released into the culture medium, which involved in the regulation of bystander effects. Some research found that radiation-induced changes of miRNA could also participate the regulation of bystander effects induction. In order to further investigation the miRNAs and its role of radiation response, in this paper, we choose the normal human bronchial epithelial cell BEP2D as the target cell, after 2Gy irradiation, the cell culture medium was collected and from which the nanoparticles exosomes were separated, the miRNA expression profile of the exosomes was analysed via miRNA microarray. We selected some radiation response exosomal miRNAs to further verify their expression changes and biological effects. We hoped the investigation about the function of radiation-related miRNA in the bystander effects can provide important reference value for the risk assessment and novel radiation protection measure research.Purpose:The aim of study was to identify the expression pattern of miRNA molecules in the exosomes from irradiated BEP2D cells, and elucidate the role and mechnisms of the radiation-induced exosomal miRNA on the bystander effects of radiation.MethodsHuman bronchial epithelial cell line (BEP2D) was employed in this study.1) The BEP2D cells were irradiated by 2Gy of 60CO y-rays. The cells were further cultured for 4hr and 8hr, and the control cells were established in parallel.The conditioned medium (CM) from the irradiated BEP2D cells and control medium from non-irradiated control cells were collected.2) The exosomes were separation and purification via ultracentrifugation.3) The exosomes were identified via transmission electron microscope and exosomal miRNA microarray analysis was performed.4) RT-qPCR was used to detect miR-7-5p and miR-1246 expression and EGFR mRNA expression.5) Cell proliferation assay was performed using CCK-8 Kit.6) Flow cytometry for cell cycle and apoptosis detection.7) Autophay and in situ hybridization response were observed through confocal laser fluorescence microscope.9) Alteration of protein expression level was detected by Western blot assay.10) Cell micronuclei were detected by cytochalasin B.53BP1 foci was detected by fluorescent immumohybridization laser confocal observation.Results1. The microRNA chip analysis indicated that miRNAs expression profiles in the secreted exosomes from irradiated cells significantly changed, there were six miRNAs upregulated and six miRNAs downregulated at 4hr post 2Gy irradiation, and there were four miRNAs upregulated and seven miRNAs downregulated at 8hr post 2Gy irradiation.2. MiR-7-5p and miR-1246 in the exosomes from irradiated cells appeared continuously increased at least from 4hr to 8hr after 2Gy irradiation. On the contrary, their expression obviously decreased in intracellular after irradiation, and the results were confirmed via RT-qPCR (p< 0.01).3. Overexpression miR-7-5p can inhibit cell proliferation of the non-irradiated human bronchial epithelial cell BEP2D (p< 0.05), and increase autophagic vacuoles formation (p< 0.05). The autophagy-related proteins LC3B, Beclinl expression increased significantly, and the degradation of p62 protein obviously increased. miR-7-5p overexpression has no effect on apoptosis induction and cell cycle.4. The miR-7-5p level detected by RT-qPCR was significantly increased in the non-irradiated human bronchial epithelial cell BEP2D after taken up the exosomes from the irradiated cells (p< 0.05).5. Exosomal miR-7-5p also inhibited cell proliferation and induced autophagy in the non-irradiated human bronchial epithelial cell BEP2D, and above changes were reversed by miR-7-5p inhibitor.6. MiR-7-5p inhibited the expression EGFR in both mRNA level and protein level. Overexpression miR-7-5p also inhibited the p-AKT and p-mTOR protein expression, which was reversed by miR-7-5p inhibitor.7. The conditioned medium from the irradiated BEP2D cells resulted in the increased yield of micronuclei in both the non-irradiated BEP2D cells and AHH-1 cells. The formation of 53BP1 foci, the biomarker of DNA double-strand break, also significantly increased in non-irradiated BEP2D cells after the treatment of conditional medium (p< 0.05).8. Overexpression miR-1246 was found to increase the yield of micronuclei in non-irradiated BEP2D cells and AHH-1 cells, and to induce 53BP1 foci in non-irradiated BEP2D cells, and which demonstrated the similar effects produced by the conditions medium.Conclusion:Radiation can significantly alter the pattern of the exosomal miRNA profile from BEP2D. A number of upregulated or downregulated miRNA molecules have been identified from the exosomes secreted from the irradiated BEP2D cells, which included the upregulated miR-7-5p and miR-1246. Exosomal miR-7-5p was found to inhibit cell proliferation possibly through inducing autophagy. MiR-7-5p did not induce cell cycle arrest and apoptosis. Exosomal miR-7-5p induced autophagy via its target EGFR and downstream the AKT/mTOR signaling pathway. It was also found that the conditional medium from irradiated BEP2D cells resulted in the bystander effect of genomic damage, which was associated with the increased miR-1246 level in the exosomes.