| Objective: Epilepsy is a common chronic disease of the nervous system,characterized by hypersynchronized abnormal firing of neurons during seizures.During epileptic seizures,the excitatory energy expenditure of a large number of neurons increases significantly,and the glycolytic pathway can quickly supply energy to brain tissue as an important cellular energy supply bypass.Studies have shown that aerobic glycolysis levels are significantly elevated during seizures,which is very similar to the Warburg effect in tumor tissue.As a non-coding RNA,circRNA regulates various physiological processes at the epigenetic level.The most common way is to bind miRNA by acting as a competitive endogenous RNA(ce RNA),reduce the level of miRNA,and release the effect of miRNA on downstream target genes.inhibitory effect.Studies have shown that circRNAs play regulatory roles through the circRNA-miRNA-m RNA network in various diseases,but less research has been done on the pathogenesis of epilepsy.CircRNA＿100290 is a circRNA that is transcribed and reverse spliced from the SLC30A7 parental gene.Studies have reported that circRNA＿100290 can affect the level of aerobic glycolysis in oral cancer.The expression level of MiR-145 was significantly changed in patients with temporal lobe epilepsy and animal models.A binding site between circRNA＿100290 and miR-145 was found through bioinformatics website prediction.ETS1 is a widely reported oncogenic transcriptional activator in various cancers.Bioinformatics website analysis predicted that there was a binding site for miR-145 in the 3’ untranslated region(3’UTR)of ETS1.Based on the above theoretical basis and analysis results,this study detected the expression of circRNA＿100290,miR-145,and ETS1 in human temporal lobe epilepsy brain tissue and normal brain tissue,and verified the binding and regulatory relationship between the three through in vitro experiments.And the regulation of circRNA＿100290 on the level of cellular aerobic glycolysis and its mechanism of action.The effects of endogenous overexpression of circRNA＿100290on seizures and epilepsy electroencephalograms of hairenic acid amygdala-ignited rat epilepsy models were investigated by in vivo experiments.To explore the molecular mechanisms underlying the development of temporal lobe epilepsy.Methods:1.Collect intraoperative temporal lobe tissue and normal brain tissue specimens from patients with temporal lobe epilepsy surgery.RT-q PCR was used to detect the expression levels of circRNA＿100290.miR-145 and ETS1 m RNA in the brain tissues of the two groups.Western blotting(Western Blot,WB)and immunofluorescence to detect the expression level of ETS1 protein,and to verify the ring structure of circRNA＿100290 by agarose gel electrophoresis.2.The human astrocyte cell line HA(HA1800 cells)was cultured,and the localization of circRNA＿100290 in the cells was determined by fluorescence in situ hybridization(FISH);according to the results predicted by the bioinformatics website,it was detected by the dual luciferase reporter gene system circRNA＿100290 and miR-145.Interaction and binding site between miR-145 and ETS1.3.HA cells were transfected with lentivirus to construct circRNA＿100290 silencing and overexpressing cell lines,RT-q PCR and WB were used to detect the changes of miR-145 and ETS1 expression levels,seahorse energy metabolometer and glucose consumption assay and lactate production assay.Effects on the level of aerobic glycolysis in cells;miR-145 silencing and overexpression cell lines were constructed by transfecting cells with miR-145analogs(mimics)and inhibitors(inhibitor),and RT-q PCR and WB were used to detect the expression level of ETS1.Changes;miR-145 mimics were transfected into circRNA＿100290 overexpressed stable cell lines for recovery experiments,and the expression level of target protein ETS1 was detected by WB to observe whether the effect of circRNA＿100290 overexpression on ETS1 could be reversed.Seahorse energy metabolism instrument and glucose consumption assay experiments and lactate production assay to test whether the effect of circRNA＿100290 overexpression on aerobic glycolysis can be reversed.4.Intracranial injection of KA in the amygdala of the stereotaxic instrument was used to construct the epilepsy model of KA-amygdala-ignited rats.RT-q PCR and WB were used to detect circRNA＿100290.miR-145 in the hippocampus of epileptic rats and normal rats..Expression levels of ETS1.5.The adenovirus vector overexpressing circRNA＿100290 was constructed,and the rat hippocampus was transfected by intracranial injection of the virus in a stereotaxic instrument,and a rat model of local overexpression of circRNA＿100290 in the hippocampus was constructed,and KA was injected into the amygdala at the same time as the transfection control group.Then the epilepsy model was constructed,the seizure grade and latency period after KA modeling were recorded,and EEG was recorded.The expression levels of circRNA＿100290,miR-145 and ETS1 in the hippocampus of rats in each group were detected by RT-q PCR,WB and immunofluorescence.Results: In human temporal lobe epilepsy brain tissue,the expression levels of circRNA＿100290 and ETS1 were significantly lower than those in normal control brain tissue,while the expression of miR-145 was significantly down-regulated in temporal lobe epilepsy brain tissue.circRNA＿100290 can directly bind to miR-145,and the expression levels of the two in temporal lobe epilepsy tissues are negatively correlated,suggesting that circRNA＿100290 may negatively regulate miR-145.In addition,the 3’UTR region of ETS1 m RNA can also bind to miR-145,and the expression levels of the two in temporal lobe epilepsy tissues are also negatively correlated,suggesting that miR-145 may inhibit the expression of ETS1 by binding to the 3’ UTR region of ETS1 m RNA.Combined with the results of the first part,in HA cells,miR-145 can negatively regulate the expression of ETS1 by binding to the3’UTR region of ETS1 m RNA.circRNA＿100290 can bind to miR-145,play the role of a "molecular sponge" through the ce RNA mechanism,adsorb miR-145,and negatively regulate the level of miR-145,thereby affecting the expression of ETS1.Overexpression of circRNA＿100290 can inhibit aerobic glycolysis in HA cells,whereas silencing circRNA＿100290 can enhance the level of aerobic glycolysis.Overexpression of miR-145 could reverse the down-regulation of aerobic glycolysis and the up-regulation of ETS1 expression caused by increased expression of circRNA＿100290,demonstrating that circRNA＿100290 regulates aerobic glycolysis through the miR-145/ETS1 pathway.In the hippocampus of the KA amygdala-ignited rat epilepsy model,the expression levels of circRNA＿100290 and ETS1 were significantly lower than those in the hippocampus of normal rats in the control group,while the expression level of miR-145 in the hippocampus of the epilepsy model group rise.This is consistent with their changing trend in the first part of human temporal lobe epilepsy brain tissue.Local overexpression of circRNA＿100290 in the rat hippocampus can alleviate epileptic seizures to a certain extent through the miR-145/ETS1 pathway,which may be achieved by inhibiting aerobic glycolysis.Conclusions: 1.The expression levels of circRNA＿100290 and ETS1 in the brain tissue of patients with temporal lobe epilepsy were down-regulated,and the expression level of miR-145 was up-regulated,and the expression levels of circRNA＿100290 and miR-145,miR-145 and ETS1 were negatively correlated.2. There is a direct binding relationship between circRNA＿100290 and miR-145,miR-145 and ETS1.3.Overexpression of circRNA＿100290 in astrocyte HA can inhibit the aerobic glycolysis of cells,while silencing circRNA＿100290 can enhance its aerobic glycolysis.4.circRNA＿100290 binds miR-145 through ce RNA mechanism,relieves the inhibitory effect of miR-145 on ETS1 expression,and inhibits the aerobic glycolysis of cells.5.The expression levels of circRNA＿100290and ETS1 were down-regulated,and the expression level of miR-145 was up-regulated in Hairen acid amygdala-ignited rat epilepsy model.6.The local overexpression of circRNA＿100290 in the hippocampus of the amygdala-ignited rat epilepsy model can alleviate epileptic seizures in rats by regulating the miR-145/ETS1 pathway. |
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