The Regulatory Mechanism Of MiR-129-2-3p On GABRA1 And The Function Of GABA_AR_α1 Glycosylation In Refractory Epilepsy

Background:The balance of inhibitory and excitatory signals is the basis of the homeostasis of the nervous system and an important guarantee to maintain the vital activities of the body,which plays an important role in maintaining the normal physiological function of the body.Various pathogenic factors and changes in internal and external environment can lead to the imbalance of nerve signal homeostasis,resulting in abnormal central nervous system excitation and lead to a series of diseases.Epilepsy is one of the most common neurological disorders,characterized by repeated attacks due to the abnormal firing of a large number of neurons in the brain.There are an estimated 70 million cases of epilepsy worldwide and about 9 million cases in China.Epilepsy disease seriously harms the health of patients and causes great economic burden to patients’families.At present,the pathogenesis of epilepsy is not clear,and there are many theories.The more widely accepted hypothesis is that the occurrence of epilepsy is controlled by epigenetic factors and gene products,and the destruction of the homeostasis of the nervous system,that is,the imbalance of inhibitory and excitatory signal transduction is an important cause of epilepsy.The most important inhibitory neurotransmitter in the brain,GABA,does its job by binding to its receptors.There are two types of GABA receptors,ionophilic（GABAA）receptors and metabolophilic（GABAB）receptors.GABAA receptors exist in 20%-50%of synapses and respond to GABA binding on a millisecond time scale by altering the conformation of chloride channels and altering their permeability,thus inhibiting mature neuronal activity.Dysfunction of this pathway can lead to disorders such as epilepsy,anxiety disorders and autism.In particular,mutations in the gene encoding theα1subunit of the GABAA receptor（GABRA1）have been identified as a pathogenic factor in juvenile myoclonic epilepsy and idiopathic generalized epilepsy.Therefore,changes in theα1 subunit of GABAA receptor(GABA_AR_α1)are crucial in the pathogenesis of epilepsy.Micro RNAs are small non-coding RNAs（mi RNAs）involved in the regulation of post-transcriptional gene expression.Mi RNAs target the 3’untranslated region（UTR）of m RNAs for transcriptional degradation or translation inhibition,thereby negatively regulating gene expression,and can also combine with promoter region to play an activation role.Bioinformatics,systematic studies and advanced quantitative proteomics have revealed that up to 60%of human m RNAs are regulated by mi RNA-induced silencing,and each mi RNA can regulate proteins by regulating corresponding genes.The underlying pathological mechanisms in the development of epilepsy and the maintenance and development of epileptic states involve mi RNAs that control multiple genes and proteins at the systemic level.Among them,specific mi RNAs can act as post-transcriptional negative regulators of gene expression and regulate neuroinflammation,neurodegeneration,neurongenesis,neuronal excitability and synaptic function in the course of epilepsy.Glycosylation is one of the most important post-translational modifications of proteins in eukaryotes.More than half of proteins undergo glycosylation,which is involved in a variety of biological processes,including cell differentiation,growth,immune response,intercellular adhesion,and intercellular signaling.Abnormal glycosylation can affect the function of multiple organ systems,and the central nervous system is the most vulnerable,including synaptic development block and abnormal signal transmission between synapses.It is also a cause of neurological diseases such as Parkinson’s,Alzheimer’s and epilepsy.In the nervous system,N-glycan can regulate brain development and synaptic growth,thus affecting synaptic plasticity,learning and memory.Ligand-gated channel proteins can act as synaptic neurotransmitter receptors,mediating neuron to neuron signaling.These receptors are usually n-glycosylated proteins,and the glycans attached to them are often able to regulate signal transduction.Previously,our research group collected samples of cerebral epileptogenic foci（FCD focal cortical dysplasia）from Liaoning Epilepsy Disease Diagnosis and Treatment Center,the Second Affiliated Hospital of Dalian Medical University,and conducted differential mi RNA screening by comparing normal brain tissue（cerebral hemorrhage cortical stomy tissue）samples with gene chip technology.It was found that mi R-129-2-3p was significantly increased and the differential expression was obvious.It is worth noting that through bioinformatics analysis,we found that mi R-129-2-3p may target binding to the 3’non-coding region of GABRA1.Purpose:In this paper,mi R-129-2-3p may target GABRA1 and regulate its expression,and elucidate the mechanism of mi R-129-2-3p and abnormal n-glycosylation of GABA_AR_α1in refractory epilepsy,so as to provide effective guidance for the diagnosis and treatment of refractory epilepsy.Methods:1.The target genes of mi R-129-2-3p were predicted by cross-database analysis,and the binding of mi R-129-2-3p to GABRA1 was proved by double luciferase assay.2.Primary rat hippocampal cells were cultured（in vitro）and KA epilepsy animal model was constructed（in vivo）.RT-PCR,Western Blot,immunofluorescence,TUNEL detection,behavioral and electroencephalogram detection were used to verify the regulation of GABRA1 by mi R-129-2-3p.However,inhibition of its expression has certain antiepileptic effect.4.Amino acid sequences and glycosylation sites of GABA_AR_α1in different species were compared in the database5.Peptide N-glycosidase F（PNGase F）was used to demonstrate that GABA_AR_α1is a glycoprotein with glycosylation site.6.RT-PCR and Western Blot were used to reveal the changes of GABA_AR_α1expression in animal model of KA epilepsy,as well as the changes of GABA_AR_α1glycosylation revealed by immunoprecipitation and lectin reaction.Results:1.Cross-analysis of multiple databases predicted 12 candidate target genes of mi R-129-2-3p,and further analysis of GABRA1（γ-aminobutyrate A receptorα1subunit gene）by GO and KEGG was the most likely target gene for mi R-129-2-3P to regulate and play an important role in refractory epilepsy.2.Dual luciferase assay showed that mi R-129-2-3p specifically binds and regulates GABRA1.3.The expression of mi R-129-2-3p in tissue samples of patients with refractory epilepsy was significantly increased.4.In primary rat hippocampal cells and KA（a widely used and recognized refractory TLE model）induced epilepsy,the expression level of mi R-129-2-3p was increased after KA induction treatment,while the change level of GABRA1 m RNA was opposite.5.In the inhibitor group（Anta-129）,the expression level of mi R-129-2-3p was decreased while the expression level of GABRA1 m RNA was increased,and the frequency and severity of seizures were reduced in the mi R-129-2-3p inhibitor group（Anta-129）.The amplitude and frequency of electroencephalogram also decreased.However,the amplitude and frequency of EEG in the inhibitor group（Anta-129）+GABRA1 si RNA group were increased.6.Neuron apoptosis increased in KA epilepsy model and decreased in inhibitor group（Anta-129）.7,gene database search（https://www.genecards.org）from different species（humans and rats）GABA_AR_α1protein properties,and confirmed it as a glycoprotein and expression glycosylation sites.8.Western blot analysis of GABA_AR_α1in rat hippocampal tissue after treatment with peptide N-glycosidase F（PNGase F）showed that the protein position of Gabaα1 was migrated.9.The expression level of GABA_AR_α1protein in the hippocampus of KA epileptic rats decreased.The agglutinin experiment showed that the expression of N-glycosylated glycans in GABA_AR_α1decreased significantly,and there were specific glycosylation changes.Conclusion:1.In Ka-induced cells and animal epilepsy models,mi R-129-2-3p can bind to its target gene GABRA1,regulate its gene expression and participate in the process of epileptogenesis,and induce neuron apoptosis.By effectively inhibiting the expression of mi R-129-2-3p,it can be used as a new antiepileptic mechanism and a potential target for the diagnosis and treatment of refractory epilepsy.2.N-glycosylated glycans of GABA_AR_α1are generally significantly decreased in KA model of animal epilepsy,with specific glycosylated changes.Further experiments may reveal its early diagnostic value in refractory epilepsy.