The Preliminary Study On The Role Of MiR-34a/Atg4B Mediated Autophagy In Radiation On Cognitive Impairment

Objective:Autophagy is an important process for phagocytosis,degradation and digestion of material components in cells.It plays an important role in maintaining intracellular homeostasis,material recycling and cell morphological integrity.It has been found that miR-34 a is an important miRNA regulating autophagy.Mi R-34 a is upregulated in the brains of Alzheimer's disease patients and animal models.Alzheimer's disease and radiation-induced brain injury have a common pathophysiological basis,e.g.neurogenic disorder.Thus we hypothesized that radiation-induced brain injury would also cause an up-regulation in autophagy-related miRNA and a reduction in autophagy level.We attempted to irradiate rats with 20 Gy of electron beams to induce cognitive impairment.Then we screened miRNA which are significantly up-regulated or downregulated by miRNA array technology,then we combined the database(Targetscan,micro RNAorg,pita)and related literature to predict the target autophagy related gene to explore the potential role of miRNA and its target genes in radiation-induced cognitive impairment.The protective effect of minocycline on the primary mouse neurons and HT-22 hippocampal neurons has been found in the previous study in our laboratory and the underlying mechanisms has been investigated in HT-22 cells.The AMPK alpha 1 is activated by X rays,leading to the activation of autophagy pathway.This autophagy is protective and Minocycline increases autophagy mediated by AMPK alpha 1 in HT-22 cells,which in turn inhibits the radiation-induced apoptosis.We hypothesized that minocycline would also increase the protective autophagy in vivo and inhibit the apoptosis of hippocampal neurons.We used the Western Blot to verify the changes in autophagy after treatment with minocycline.Methods:Single 20 Gy whole brain irradiation(WBI)was given using 4 Me V electron beams to induce cognitive impairment,then rats were decapitated 1 months after whole brain irradiation.The hippocampus of the control group and the irradiated group were quickly dissected from the brain then cut into 1 mm3.After fixing,embedding,dehydration and drying,the autophagic body was observed using electron microscope.The hippocampus of rats was isolated and significant changes in miRNA were screened out by miRNA array.Real-time polymerase chain reaction(RT-PCR)was used to verify the changes in the expression of miRNA in the irradiated groups and control groups,then we combined the database(Targetscan,micro RNAorg,pita)and related literature to predict the target autophagy related gene,and the level of the target gene was verified by Western Blot.In the second prat 36 male Sprague-Dawley rats one month old were divided into four groups including control(CN),minocycline only(CM),irradiation(RN),irradiation combined with minocycline(RM).Minocycline fed by 90 mg/kg before and 45 mg/kg daily after irradiation.Single 20 Gy whole brain irradiation(WBI)was given using 4 Me V electron beams.Hippocampus tissue was separated from brain after one month of whole brain irradiation,and the protein level of LC3 II and P62 was detected by Western Blot.Conclusion:We observed autophagy in the hippocampus of rats 4h after whole brain irradiation.One month after whole brain radiation,the expression of miR-34 a in the hippocampus increased and the protein expression of Atg4 B decreased,miR-34a-targeted Atg4 B is an important regulatory gene for autophagy.We hypothesize that the autophagy mediated by miR-34a/Atg4 B may play an important role in radiation-induced cognitive impairment.In addition,irradiation may lead to long-term downregulation of autophagy in hippocampus,while minocycline can promote autophagy.