CircRNA SRRM4 Regulates The Alternative Splicing Of PKM And Promotes Aerobic Glycolysis Via Deubiquitination Of SRSF3 In Epilepsy

Objective:Epilepsy is a disease with complex pathogenesis.In recent years,although with the rapid development of antiepileptic drugs,nearly one-third of patients will develop drug-resistant epilepsy.In the intermittent period of epileptic seizures,epileptic lesions often appear as hypometabolism areas on ¹⁸F-FDG-PET,while they appear as obvious hypermetabolism areas during seizures.This phenomenon is an important method to guide the localization diagnosis of epilepsy in clinic.Interestingly,some study found that this abnormal phenomenon of glucose metabolism may not only be a manifestation of epilepsy,but also an important pathophysiological mechanism to promote epilepsy.Ketogenic diet is an energy replacement therapy,which converts the patient’s energy source from glucose to ketone body.It has achieved good results in the treatment of drug-resistant epilepsy.Moreover,experimental studies have found that the treatment of lactate dehydrogenase（LDH）has also achieved the effect of inhibiting epilepsy,making the glucose metabolism mechanism of epilepsy a research hotspot.Noncoding RNA plays an important role in many diseases,and its role in epilepsy has also been widely studied in recent years.Noncoding RNA includes long noncoding RNA（lnc RNA）,micro RNA（mi RNA）and circular RNA（circRNA）.CircRNA has attracted more attention in recent years because of their more stable structure and high abundance expression in brain tissue.Many studies have found that some circRNA promotes neuronal damage and apoptosis in epilepsy.Our study intends to further explore the mechanism of circRNA involved in regulating glucose metabolism in neurons and astrocytes in epilepsy.Methods:1.Total RNA was extracted from the focal brain tissue of human temporal lobe epilepsy（TLE）patients and normal control brain tissue for q RT PCR to verify the abnormally expressed circRNA;2.The rat model was established by injecting kainic acid（KA）into the amygdala.After taking samples in different stages of epilepsy（acute stage,plateau stage and chronic stage）,q RT-PCR,fluorescence in situ hybridization（FISH）and immunofluorescence（IF）were performed to verify the abnormally expressed circRNA according to different regions（whole hippocampus,CA1 region,CA3region and DG region）;3.The seizures neuronal cell model of SY5Y cells was induced by simulating different concentrations of KA.The abnormally expressed circRNA was verified by q RT-PCR,and the effect of different concentrations of KA on cell viability was proved by MTT assay;4.After Sanger sequencing,RNase R and actinomycin D treatment,q RT-PCR,agarose electrophoresis,FISH and nucleocytoplasmic isolation PCR were carried out to verify the circular structure and expression distribution of the circRNA;5.The expression of PKM1、PKM2 in brain lesions and normal human brain tissues were verified by q RT-PCR and western blot;6.In the rat model of TLE,¹⁸F-FDG-PET-CT was performed at different stages of epilepsy（acute stage,latent stage and chronic stage）,and the changes of glucose metabolism uptake in rat brain were observed;7.In the rat model of TLE,after taking samples in different stages of epilepsy（acute stage,latent stage and chronic stage）,q RT-PCR and Western blot experiments were carried out to different regions（whole hippocampus,CA1 region,CA3 region and DG region）to verify the expression levels of PKM1 and PKM2,and the IF experiment was double stained PKM2 with Neu N and GFAP to observe the expression distribution;8.After adding KA to SY5Y and NHA cells to simulate epilepsy cell model,ECAR,OCR,glucose uptake rate and lactate production rate were perform to detect their effects on cell glucose metabolism;9.Overexpression or silencing circSRRM4 by lentivirus transfection in SY5Y and NHA cells,and the transfection efficiency was verified by q RT-PCR;10.After overexpression or silencing circSRRM4 in SY5Y and NHA cells,q RT-PCR and Western blot experiments were carried out to verify its effects on the transcription and expression of PKM1 and PKM2;11.After overexpression or silencing circSRRM4 in SY5Y and NHA cells,ECAR,OCR,glucose uptake rate and lactate production rate were tested to detect their effects on cell glucose metabolism;12.Circs SRRM4 was silenced by in vivo transfection of adeno-associated virus（AAV）in rat hippocampus.GFP fluorescence efficiency was observed in frozen sections,and q RT-PCR to verify the transfection efficiency and specificity;13.Two weeks after silencing circSRRM4 by AAV transfection,the epilepsy model was established,and then the seizure frequency,EEG monitoring and¹⁸F-FDG-PET-CT examination were observed during the chronic seizure period to observe the effect of silencing circSRRM4 on epilepsy and glucose metabolism in brain tissue;14.After silencing circSRRM4 by AAV transfection,the rat model was taken samples after the completion of the in vivo experiment of chronic stage,the q RT-PCR and Western blot experiments were carried out to verify the effect of silencing circSRRM4 on the transcription and expression of PKM1 and PKM2 in vivo;15.After silencing circSRRM4 by AAV transfection,the rat model was taken samples after the completion of the chronic stage in vivo experiment.The effects of silencing circSRRM4 on PKM2 and activated astrocytes were observed by IF double staining PKM2 and GFAP,and the effects on neuron loss were observed by Nissl staining;16.Add biotin labeled circSRRM4 probe to the cell lysate stably transfected with overexpressed circSRRM4 for pulldown experiment.After SDS/PAGE,silver staining was carried out to analyze the differential bands,and the bands were cut off for mass spectrometry（LC-MS）to identify the binding protein;17.The transcription and expression of SRSF3 were verified by q RT-PCR and Western blot in the focal brain tissue of human TLE patients and normal control brain tissue;18.After overexpression or silencing circSRRM4 in SY5Y and NHA cells,q RT-PCR and Western blot experiments were carried out to verify its effect on SRSF3transcription and expression;19.The binding of circSRRM4 to SRSF3 was verified by RNA immunoprecipitation（RIP）and pulldown experiment;The binding effect of circSRRM4 and SRSF3 was verified by FISH and IF double staining in SY5Y and NHA cells;20.In the cells overexpressing or silencing circSRRM4,CHX or MG132 was added to the cells,and then western blot was carried out to observe the regulatory effect of circSRRM4 on the post transcriptional expression of SRSF3.Immunocoprecipitation（IP）was carried out by SRSF3 antibody,and Western blot was carried out by ubiquitin antibody to verify the effect of circSRRM4 on the ubiquitination level of SRSF3.21.By silencing SRSF3 in cells with overexpressed circSRRM4,and then performing q RT-PCR,Western blot,OCR,ECAR,glucose uptake rate and lactate production rate,to verified that circSRRM4 regulates the alternative splicing process of PKM through SRSF3,thereby affecting the disease phenotype.Results:1.The expression of circSRRM4 in brain tissue of TLE patients was significantly increased;2.Compared with the control group,the expression of circSRRM4 increased significantly in the acute phase,then returned to normal in the latent phase,and increased again in the chronic phase;The expression of circSRRM4 was significantly increased in CA3 region and DG region in epilepsy group,but there was no significant difference in CA1 region;3.The expression of circSRRM4 was significantly increased after Add 200-250μM KA to SY5Y cells;4.CircSRRM4 can exist in a stable circular structure and is mainly distributed in the cytoplasm;5.The hippocampal tissue of epileptic rat model showed abnormal glucose metabolism:the glucose uptake rate of hippocampal lesions on KA injection side significantly increased in the acute stage,decreased in the latent stage,and decreased most significantly in the chronic stage;6.Through FISH and IF double staining experiments,it was found that circSRRM4was mainly expressed in the cytoplasm of hippocampal cells in the control group and showed weak fluorescence intensity.The fluorescence intensity of circSRRM4increased significantly in the acute phase,and returned to a lower level in the latent phase,Finally,in the chronic phase,it was found that the fluorescence intensity of circSRRM4 in CA3 region increased significantly,and the co localization index with GFAP increased significantly at the same time;7.In TLE patients,the transcription and expression of PKM1 decreased significantly,while the transcription and expression of PKM2 increased significantly;8.The transcription level of PKM2 increased significantly in the acute phase of epilepsy and decreased slightly in the latent phase.After entering the chronic phase,the transcription level of PKM2 increased significantly again.The transcription level of PKM1 increased significantly in the acute phase of epilepsy,decreased to the normal level in the latent phase,and decreased significantly in the chronic phase;9.In the acute phase of epilepsy,the expression of PKM2 in hippocampal pyramidal cells increased significantly,decreased to the normal level after the latent phase,and increased again after the chronic phase.GFAP began to rise when entering the latent phase,and increased significantly after the chronic phase.Through co localization analysis,it was found that the co localization index of PKM2 and GFAP began to increase significantly after entering the latent stage to the chronic stage;10.Silencing circSRRM4 can reduce the seizure frequency,improve the local glucose metabolism and prevent the hippocampal neuron loss in epileptic rats,reduce the transcriptional expression of PKM2 and alleviate the activation of astrocytes;11.CircSRRM4 can promote the transcription and expression of PKM2 and inhibit the transcription and expression of PKM1 in SY5Y and NHA cells;CircSRRM4 can promote glycolysis,glucose uptake and lactate production in SY5Y and NHA cells,and reduce aerobic phosphorylation;12.CircSRRM4 can combine with SRSF3 and inhibiting SRSF3 from entering the ubiquitin proteasome system,so as to increase the expression of SRSF3;13.CircSRRM4 can regulate the alternative splicing process of PKM through SRSF3,promote the transcription of PKM2,and then affect the phenotype of the disease.Conclusions:1.CircSRRM4 can increase the stability of SRSF3 by reducing its ubiquitination level,so as to regulate the alternative splicing process of PKM,improve the expression of PKM2,improve the glycolysis level of cells,and provide energy basis for the pathogenesis of epilepsy;2.Silencing circSRRM4 can reduce the incidence rate and seizure frequency of epilepsy,improve the local hypometabolism of hippocampus,protect the neurons and alleviate the activation of astrocytes.