Alteration And Mechanism Of Exosomal MiRNAs In The HUVEC Response To Ionizing Radiation-induced Injury

Objective:To identify the expression pattern of miRNAs in intracellular and exosomes of human umbilical vein endothelial cells?HUVEC?after irradiation,and the relationship of miRNAs expression changes with time post-irradiation.The conditional culture medium of irradiated cells was used to explore the biological effects and mechnisms of radiation-induced bystander effect?RIBE?on HUVEC cells.Methods:?1?The HUVEC cells were irradiated by 4Gy of ⁶⁰Co?-rays and then incubated for 0.5h and 2h,and the control cells were established in parallel.The radiation-induced bystander effect?RIBE?was investigated by a medium transfer method[irradiated cells conditioned medium?ICCM?transfer].ICCM from HUVEC cells was collected 0.5h or 2h after irradiation and filtered through a 0.22?m filter and control cell conditioned medium?CCCM?from non-irradiated cells was collected in parallel.?2?The exosomes were separated and purificated from the collected ICCM or CCCM via ultracentrifugation.?3?The exosomes were identified by transmission electron microscope?TEM?and the miRNA microarray technique was used to analyze the extracellular and mi RNAs in the exosomes.?4?Real-Time PCR was used to validate the significance of the differentially expressed miRNAs.miRDB and TargetScan were performed to predict the target genes of thedifferentially expressed miRNAs.DAVID,KEGG and other online tools were used for bioinformatics analysis.The Cytoscape software was used to construct the protein–protein interaction?PPI?network for key genes analysis.?5?Cell proliferation was detected by CCK8 Kit.?6?Flow cytometry was used for apoptosis,cell cycle and ROS levels detection.?7?Cell micronuclei were detected by cytochalasin B.Fluorescent immumohybridization laser confocal observation was performed to observe 53BP1 foci and the intake of exosomes.?8?Using Western blot assay to detect related proteins expression level.Results:?1?mi RNA microarray analysis revealed that mi RNAs expression profiles in intracellular and exosomes significantly changed after 4Gy of⁶⁰Co?-ray irradiation.Compared with the control exosomes,five up-regulated,thirteen down-regulated miRNAs were identified at 0.5h post 4Gy-irradiation,and sixteen upregulated and five downregulated miRNAs at 2h post 4Gy-irradiation.Moreover,thirty-eight miRNAs and eighty-five miRNAs were significantly differentially expressed respectively in intracellular at 0.5h and 2h after radiation?P<0.01?.Especially,mi R-6729-5p in the exosomes from irradiated cells appeared continuously increased at least from 0.5hr to 2hr after 4Gy irradiation.?2?A portion of differentially expressed mi RNAs were confirmed via RT-qPCR?P<0.05?.It was found that miR-4267,miR-5096,miR-1307-3p,miR-1260b,mi R-6729-5p,miR-4638-5p,let-7e-5p et al were significantly upregulated in intracellular after 0.5h irradiation?P<0.05?.Yet,only five miRNAs were identified in intracellular after 2h irradiation?P<0.05?,they are miR-29b-3p,mi R-5096,mi R-615-5p,miR-1307-3p,miR-1260b and mi R-6729-5p.?3?KEGG analysis showed that the target genes of the up-regulated miRNAs were majorly enriched in MAPK pathway,endocytosis,Rap1pathway,Ras pathway,Hippo pathway,while the target genes regulated by down-regulated miRNAs were primary enriched in axon guidance,cAMP pathway and so on.?4?Based on the information of bioinformatics analysis and published papers,it was believed that YWHAZ may be the target gene to miR-6729-5p,miR-5096 is likely to target BRCA1,HDAC1,TGFBR1,PIK3R1,UBQLN1,and miR-1260b probably related with YWHAZ,SMAD2 PTPN11 and so on.The above miRNAa seem to participate in the regulation of cell radiation injury reaction or radiation side effects,which involved in the regulation of radiation response or RIBE.?5?Iirradiated cell conditioned medium?ICCM?was found to inhibit cell proliferation?P<0.05?,increase apoptosis induction?P<0.01?and ROS level,and result in the increased yield of micronuclei?P<0.01?.Meanwhile,the formation of 53BP1 foci,a biomarker of DNA double-strand break,was significantly increased in HUVEC cells after the treatment of ICCM even without irradiation.?6?RIBE of G2/M cycle arrest was induced in non irradiated cells by ICCM.A similar trend was observed in Western Blotting and flow cytometry.Conclusion:Radiation can significantly change the miRNAs expression profiles in intracellular and exosomes,which occurs in time dependent manner.A series of upregulated or downregulated miRNAs have been confirmed via RT-qPCR,especially the upregulated miR-6729-5p.YWHAZ was predicted to be a main target of miR-6729-5p,mi R-5096 might target HDAC1,TGFBR1,PIK3R1,BRCA1 and UBQLN1,and mi R-1260b might target SMAD2,PTPN11,YWHAZ,UBQLN4 etc.The identification of differentially expression miRNAs provided further information for the elucidation of the regulation mechanisms of radiation response or RIBE.Anyway,ICCM was found to inhibit cell proliferation and increase apoptosis induction?P<0.01?.ICCM also resulted in genomic damage in bystander cell,which was demonstrated by the increased yield of micronucle i?P<0.01?and the formation of 53BP1 foci.Most importantly,RIBE led to G2/M cycle arrest in non irradiated cells,possibly through activing CHK1-pS345/CHK2-pT68.