Study On The Phenomenon And Mechanism Of Golgi Dispersion In Radiation-Injured Cells

The Golgi apparatus is an important organelle in endomembrane system,involved in secretion,vesicle transport andintracellular protein process.Recent years,it has been found that DNA damage can lead to the reorganization of the Golgi apparatus,which is fragmented and dispersed into the whole cell.Although the results were published in the journal Cell,the mechanistic information on this field is very limited.Whether the structural change of the Golgi apparatus really involves in cell survival,or is merely a reaction with the cell lesions,this paper will address this question,to investigate the dispersion of Golgi apparatus after radiation injury and its significance and mechanism.PurposeTo verify the dose-and timen-effects of ionizing radiation on the morphology of Golgi apparatus,to investigate the mechanism of the diffusion of Golgi apparatus and biological significance.MethodUsing immunofluorescence technique to observer the Golgi dispersion in radiation-injuryed cells,and dispersion statistics area was measured.The cell cycle was determined by flow cytometry,cell survival analysis was performed by cell colony forming experiment.Western Blot was used to detect the expression of GOLPH3 and DNA damage marker ?H2AX,the changes of miR-378 e and miR-486-3p expression and the expression of target moloeculesGOLPH3 L and MYO18 A were detected by RT-PCR.ResultImmunoflurescence observation showed that the golgi dispersal caused by 60 Co ?–ray was significantly increased in adose-dependent manner in the range of 4Gy ~10Gy.This significantphenomenon was demonstrated by the fact that the golgi area was significantly increased.When the irradiated cells were treated with the radioprotective agent VND3207,the golgi dispersal induced by radiation was significantly reduced.This radiation-induced golgi dispersal was also displayed in a pattern of time-course after irradiation in the He La cells,and persisted at least to 36 h post-irradiation.Cell cycle test results indicated that the golgi dispersal was not associated with the G2/M arrest triggered by radiation-induced DNA damage response.VND3207 can promote cell survival by colony formating assay.The results of RT-PCR showed that after 4Gy or 10 Gy irradiation,in the expressions of miR378 e,miR 486-3p,GOLPH3 L and MYO18 A were upregulated in HUVEC cells.immunofluorescence assay showed that transfection of miR 378 e,miR 486-3p,siGOLPH3 L cells resulted in the Golgi apparatus diffus,and transfection of siGOLPH3 L and siMYO18 A could alsoprolonged the G2/M blockage induced by irradiation.Finally,transfection of miR 378 e,miR486-3p,siGOLPH3 L,siMYO18A increased the sensitivity of cells to radiation.Conclusion1 Ionizing radiation obviously induced the the Golgi apparatus dispersion,and which displayed with the dose effect and time effect.2 Although it was reported that golgi dispersion might be related to the cell cycle G2/M block,G2/M arrest could not be a mechanistic cause for golgi dispersion induced by radiation.3 The diffusion of the Golgi apparatus was related to the degree of cell injury,and which was inhibited by the radiation protective agent VND3207,a vanillin variant with the effect of activating DNA-PKcs.4 NU7441,an inhibitor of DNA-PKcs,inhibited the repair of DNA damage,was also along with the inhibition of the golgi dispersion.5 The key protein of golgi structure regulation GOLPH3 was down regulated by irradiation,and could be down regulated by DNA-PKcs knockdown.6 miRNAs miR-378 e ? miR-486-3p might play an important role in the radiation-induced diffusion of golgi apparatus.