The Study On The Role Of Hamartin And Its DNA Methylation In Hypoxia Preconditioning Neuroprotection

Objective To investigate the role of Hamartin and its DNA methylation in hypoxic preconditioning neuroprotection by replicating hypoxic preconditioning mouse models and establishing hypoxic preconditioning models in vitro.To explore the role of Hamartin in hypoxia and whether it plays a role under hypoxic preconditioning is caused by its DNA methylation.Methods 5-aza-cd R was injected into the cerebral ventricles of mice,and then hypoxic treatment was performed.The mice were sacrificed according to the requirements.The hippocampus was isolated and the hippocampal CA1 and CA3 regions were isolated.Transcriptional levels of DNA methyltransferases DNMTs and Hamartin in hippocampus CA1 regions and CA3 regions were detected by Q-PCR;Western blot was used to detect the expressions of Hamartin in hippocampus CA1 regions and CA3 regions.Detect the methylation sites in the promoter region in hippocampus CA1 regions and CA3 regions;The induced HT22 cells were treated with hypoxia and hypoxia preconditioning.The changes of cell morphology were observed by inverted microscope and confocal laser scanning.The m RNA expression of DNMTs and Hamartin were detected by Real-time PCR The changes of the methylation sites in the promoter regions in different treatment groups were detected.The changes of the luciferase reporter gene activity in different treatment groups were detected.Flow cytometry was used to detecte cell apoptosis.Results(1)There was no significant difference in Hamartin expression between the BSA-treated group and the5-aza-cd R-treated group.(2)The hippocampal CA1 and CA3 regions of the mouse were isolated.The levels of Hamartin and DNMTs in the CA1 and CA3 regions were different after hypoxic treatment.Changes in Hamartin,DNMT1,and DNMT3 B were similar in the BSA-treated and 5-aza-cd R-treated groups.(3)The expression of Hamartin in CA1 and CA3 regions was different in mice.After hypoxic treatment,the expression of Hamartin protein in CA3 was increased.(4)After HT22 cells were induced,the transcription level of Hamartin and DNMTs was significantly increased.After transfected with TSC1 plasmid and treated with hypoxia,it was found that early apoptosis of cells was significantly reduced and Hamartin may play a protective role.(5)Analysis of luciferase reporter results showed that the promoter activity in the hypoxic group wasdecreased,but the activity in the hypoxic preconditioning group was increased.(6)Analysis of sequencing results showed that the methylation sites of mouse hippocampal promoter region did not change in animal tissues.In HT22 cells,the methylation sites of promoter regions No.2 and No.22 changed in the hypoxic preconditioning group.Conclusions The neuroprotective effect of Hamartin under hypoxic conditions may be affected by the regulation of methylation of its promoter region.