The Mechanisms Of Synaptic Plasticity Regulated By MicroRNA-134 In The Development Of Epilepsy

As a common disease of neurlogy,the pathogenesis of epilepsy is still unkown.According to current studies,in addition to genetic and environment factors,synaptic plasticity,abnormal ion channel,neuron apoptosis,trauma and inflammation are likely to be the cause of epilepsy.Above all,synaptic plasticity which based on mossy fiber sprouting is the common basis of many pathophysiological processes of epilepsy.The recurrent episode of epilepsy leads to synaptic remodeling and abnormal network structure forming,finally promoting the recurrent seizures and developing to refractory epilepsy.miRNAs can regulate multiple links of epileptic development at postranscriptional level,and there are many miRNAs express abnormally in epilepsy.This study focus the changements of the expression of miRNAs in patients with epilepsy by using high-throughput sequencing technologies,screening differentially expressed miRNAs,predicting target genes by bioinformatics technologies,and then validating the relationships by dual luciferase reporter assay.Finally,exploring the specific mechanism of miR-134 regulate the development of epilepsy both in vivo and in vitro,and providing a theoretical basis for the study of the etiology of epilepsy and the development of antiepileptic drugs.Objective:1.To exploring the changes of the expression of miRNAs in the brain of patients with epilepsy and then screening the differential expression miRNAs in epilepsy and predicting target genes.2.To exploring the rat primary hippocampal neurons in vitro culture method and Identifiying the neurons.To constructing miR-134 mimics and CREB,SYT11,MAPT dual luciferase reporter gene vectors.Using dual luciferase reporter gene system to verify the relationship between miR-134 and CREB,SYT11,MAPT.3.To over-express and suppress miR-134 in vitro and in vivo epilepsy models,detecting the expression of CREB,SYT11,and analyze the mechanisms.Methods:1.To sequencing the non-coding sequences of hippocampus of patients with epilepsy and normal temporal lobe tissues by using high-throughput sequencing technologies.Comparing with known database,then identifying the known miRNAs and predicting novel miRNAs.To screening the differential expression miRNAs in epilepsy by standardized analysis,and predicting target genes by bioinformatics technologies.2.Extracting the primary hippocampal neurons from Wistar fetal rats of different gestational ages,and comparing the neurons at different growth densities.To Identifiying the rat primary hippocampal neurons cultured in vitro with neuron-specific antibody MAP-2 by immunocytochemistry and immunofluorescence.Designing miR-134 overexpression vector through the miRBase database,CREB,SYT11,and MAPT dual luciferase reporter gene vectors through the UCSC database and the NCBI database.Using dual luciferase reporter gene system to detect target genes luciferase activity.3.The epilepsy cell model was constructed by non-magnesium extracellular fluid,and the chronic epilepsy model was induced by pentylenetetrazol.Based on miRBase database,miR-134 overexpression,interference sequences were designed,then used recombinant adenovirus as vector to infect epilepsy cells and epileptic rats.Real-time fluorescence quantitative PCR detected the mRNA expression of miR-134,CREB,SYT11 in vivo and in vitro,western blot detected the protein expression of CREB,SYT11.Results:1.After all datas were filtered,1206,1196 and 1202 known miRNAs were identified in 3 samples of the epilepsy group.48,59,and 55 novel miRNAs were predicted.1056 known miRNAs were identified and 53 novel miRNAs were predicted in the control group.After normalized analysis of the expression,38miRNAs were found to be up-regulated in hippocampus of patients with epilepsy,of which 31 were known miRNAs and 7 were novel miRNAs;17 miRNAs were down-regulated,among which 11 were known miRNAs and 6 were novel miRNAs.15,700 target genes were predicted for the differentially expressed 2701 miRNAs,including CREB,SYT11,MAPT and so on.2.The average purity of neurons extracted from E16 fetal rat hippocampal tissue was 96.1%,and the average purity of neurons extracted from E17 fetal rat hippocampus was 95.3%,the average purity of neurons extracted from E18 fetal rat hippocampus was 94%,the average purity of neurons extracted from E19 fetal rat hippocampus was 90.6%.Hippocampal neurons extracted from E16 fetal rat hippocampus grew at a density of 5×10~5/ml to 2×10~6/ml in serum-free medium with B27 supplement with high nerve purity and grew stably.Using MAP-2 as the neuron-specific antibody,the deposition of brown-yellow particles in the extracted neuronal cells can be observed by immunohistochemistry;green fluorescence can be seen at the neuronal cell bodies and axons from fluorescence microscopy by immunofluorescence.The fluorescence of miR-134+CREB WT was significantly lower than that of miR-134+CREB mut.The fluorescence of miR-134+SYT11 WT was significantly lower than that of miR-134+SYT11 mut(binding site 1)(P<0.05);There was no significant difference in the fluorescence between miR-134+SYT11 WT(binding site 2)and miR-134+SYT11mut(binding site 2)(P>0.05);There was no significant difference in the fluorescence between miR-134+MAPT WT and miR-134+MAPT mut(P>0.05).3.Both in the in vitro and in vivo models of epilepsy,the expression of miR-134 decreased and the expression of CREB and SYT11 increased.The expression of CREB and SYT11 can be decreased by overexpressing miR-134,and the expression of CREB and SYT11can be increased by suppressing miR-134.Supressed the expression of miR-134 can alter the synaptic morphology of neuronal cell,increase dendritic spine density,and the frequency and extent of seizures of the epileptic rats can also been increased.Conclusion:1.The expression of miRNAs in the hippocampus of patients with epilepsy was changed,and the expression of miRNAs was mainly up-regulated.2.The rat primary hippocampal neurons can grow stably with high neuron purity at a density of5×10~5/ml-2×10~6/ml in serum-free medium with B27 supplement.3.CREB and SYT11 are target genes of miR-134.miR-134 inhibits the expression of CREB and SYT11.4.In the in vitro and in vivo model of epilepsy,miR-134 affects the development of epilepsy by regulating synaptic plasticity related genes CREB and SYT11.