Differential Expression Profiles Of MicroRNA Induced By Ionizing Radiation And Its Regulating In Radiation Damage Response

Objective:To investigate the differential expression profiles of microRNA in tritiated water and ⁶⁰Coγ-ray irradiated mice, to screen specificity miRNA of ionizing radiation. To explore their main functions in mediating hepatic response to ionizing radiation and the effects of the miR-34a on the cell cycle of BRL which were induced damage by ⁶⁰Coγ-ray. These results can provide experimental basis for further research on mechanism of miRNA regulating radiation induced damage, and promote exploring miRNA as the means of damage treatment.Methods:1.SPF C57BL/6J mice were exposured to tritiated water at different initial concentrations and were sacrificed at different time. Total number of peripheral WBC and the fMNPCE were measured. miRNA microarray was carried out to investigate the differentially expressed miRNA in liver. Some miRNAs were confirmed by Real-Time PCR assay. Bioinformatic analysis was applied to explore target genes and the main functions of the differentially expressed miRNA.2.SPF C57BL/6J mice were exposured to single whole body radiation ⁶⁰Coγ-ray at 4Gy. Total number of peripheral WBC and the fMNPCE were measured on the 3rd day. miRNA microarray was performed to investigate the differentially expressed miRNA in liver. miRNA-124 was confirmed by Real-Time PCR assay. Bioinformatic analysis was applied to explore target genes and the main functions of the differential expressed miRNA.3.Rat BRL cells were exposured to 4Gy of ⁶⁰Coγ-ray, and then respectively cultured at different time. Flow cytometry was applied to detect the changes of BRL cell cycle. Real-Time PCR assay was applied to detect the expression of rno-miR-34a and c-myc gene mRNA. P53 and Myc protein were detected by Western blot assay.Results:1.At the same initial doses, the total number of peripheral WBC decreased before the 6th day and started to recover from day 10 to day 28. The fMNPCE increased conti- nuously to the 28th day. Moreover, the total number of peripheral WBC decreased, and the fMNPCE in bone marrow increased in a dose-dependent fashion. The exposure of tritiated water resulted in many miRNAs differentially expressed: there were 20 up- regulated and 21 down-regulated in the group of exposure at the 2d(5.55×10⁵Bq/g); 9 up-regulated and 11 down-regulated in the group of exposure at the 10d(5.55×10⁵Bq/g); 32 up-regulated and 34 down-regulated in the group of exposure at the 10d(16.65×10⁵ Bq/g). mmu-miR-124, mmu-miR-200b, mmu-miR-705 and mmu-miR-214 play key ro- les in regulating target genes mediating the tritiated water-induced response. GO analys- is showed that some pathways were activated by miRNA, including MAPK ,TGF-β,VE- GF and other signaling pathways. The expression levels of specific miRNAs were accordant with the result of Real-Time PCR assay. Change between the expression of mmu-miR-34a and myc mRNA was observed a negative correlation.2. The total number of peripheral WBC decreased, while the fMNPCE in bone marrow increased after 4Gy ofγ-ray irradiation. miRNA microarray revealed that 17 miRNAs were differentially expressed, including 9 up-regulated and 8 down- regulated miRNAs. mmu-miR-34a,mmu-miR-124,mmu-miR-382 and mmu-miR-92a* play key roles in regulating target genes mediating the response to radiation. GO analysis showed that some pathways were activated by miRNA , including MAPK and B/T cell receptor signaling pathway. The expression level of miR-124 was accordant with the result of Real-Time PCR assay. There was a negative correlation between the expression of mmu-miR-34a and myc mRNA.3.After 4Gy ⁶⁰Coγ-ray irradiation , G₂ cell cycle arrest appeared at 4h and restored at 24h; the population of S cells decreased at 4h and G₀/G₁ cell cycle arrest appeared, which proceeded to 48h. The expressions of P53 protein and rno-miR-34a mRNA increased at 4h, decreased at 12h and restored at 24h after irradiation, the expression of c-myc decreased continuously in a time-dependent manner(4-24h) and restord to the level of the control at 48h. Conclusions:1.Exposure to tritiated water and ⁶⁰Coγ-ray in mice resulted in some miRNAs differentially expressed in their livers. The change of miRNA was affected by some factors including irradiation types,doses,exposure time and others. Different signaling pathways were activted by tritiated water and ⁶⁰Coγ-ray respecitively.2.miR-34a may mediate the target gene c-myc in mediating damage response to ionizing radiation in vivo.3.After radiation damage, P53/miR-34a/myc signaling pathway may modulate DNA repair by inducing cell cycle arrest in vitro.