Bystander DNA Damage Induced By Exosomes-packaged MiR-1246 From Irradiated Cells And Its Mechanism

Background and AIM: An increasing number of studies have recently reported that microRNAs(miRNAs)packaged in exosomes contribute to multiple biological effects such as cancer progression;however,little is known about its role in the development of radiation-induced bystander effects(RIBEs).In order to understand the role of exosomes in the process of RIBEs,this study was designed to identify expression change patterns of miRNAs molecules in the exosomes secreted from irradiated normal human cells,and their effect on genemoic DNA damage of receipt non-irradiated cells and the related mechanism.Methods: The exosomes were isolated from the culture medium of human bronchial epithelial(BEP2D)cell with or without 2Gy of ?-rays irradiation by ultraspeed centrifugation.Cells proliferation was analysed by CCK-8 assay and colony forming ability.DNA double-strand break(DSB)biomarker53BP1 foci detection,comet and micronucleus assays were performed for monitoring the DNA damage in the cells treated with exoaomes,or miR-1246 mimics,irradiated cells conditional culture medium(RCCM).The DSB non-homologous end joining(NHEJ)efficiency,DNA repair genes expression,and miR-1246 targeting sequence on DNA ligase4(LIG4)mRNA 3'UTR were assessed to investigate the mechanism ofbystander genomic DNA damage.Results: A set of miRNAs molecules were identified,by the miRNAs microarray analysis,to be significantly induced in exosomes isolated from the culture mediun of 2 Gy irradiated BEP2 D cells,among which there included the miR-1246 molecule.The increased expression of miR-1246 was further confirmed in the exosomes secreted by 2Gy-irradiated BEP2 D cells,and miR-1246 displayed an inhibition on the proliferation of non-irradiated cells.Either exosomes from irradiated cells,or RCCM and miR-1246 mimics obviously induced the yield of DNA DSBs in terms of increased levels of 53BP1 foci,micronuclei and comets in the non-irradiated cells.This bystander genomic DNA damage effect by exosomes or RCCM was significantly attenuated by miR-1246 inhibitor or removal of exosomes from the RCCM.miR-1246 inhibited NHEJ repair efficiency,and which was associated with the depressed expression of LIG4,a key component of NHEJ pathway.A sequence targeted by miR-1246 was further confirmed in the 3'UTR of LIG4 mRNA.Conclusion: Our study demonstrated that miR-1246 packaged in exosomes mediates the bystander DNA damage in non-targeted BEP2 D cells,and that this effect was associated with the suppression of the DSB NHEJ repair pathway through targeting LIG4.Thus,exosomal-derived miRNAs may be a critical predictor and player of the bystander DNA damage.