Genome-wiide Microrna Profiling Of Rat Hippocampus After Status Epilepticus Induced By Amygdala Stimulation Identifies Modulators Of Neuronal Apoptosis

ObjectiveEpilepsy is a common neurological disorder, affecting almost0.5%to1%of thepopulation. It is often associated with hippocampal sclerosis, which is histopathologicallycharacter-ized by selective neuronal cell loss, gliosis and synaptic reorganization.MicroRNAs (miRNAs) are small and endogenously expressed non-coding RNAs thatnegatively regulate the expression of protein-coding genes at the translational level. Emergingevidence suggests that miRNAs play critical roles in central nervous system underphysiological and pathological conditions. However, their expression and functions in statusepilepticus (SE) have not been well characterized thus far. In the current study, by usinghigh-throughput sequencing, we investigated the miRNA expres-sion profiles in rathippocampus after SE induced by amygdale stimulation. In addition, the association betweendifferentially expressed miRNAs and neuronal apoptosis was also evaluated in this study.Methods1、Here, by using high-throughput sequencing, we characterized miRNA expressionprofile in rat hippocampus at24hours following SE induced by amygdala stimulation. Weselected miRNAs that met the following criteria for additional qRT-PCR (Real-timeQuantitative Reverse Transcription Polymerase Chain Reaction) analysis:(a) having at least30copies in either experimental or control group (24hours post-SE).(b) Showingfold-change (log2)>2or fold-change (log2)<-2between these two groups (P<0.01and P≠0).2、After confirmation by qRT-PCR, six miRNAs were found to be differentiallyexpressed in brain after SE.3、Subsequent KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysisindicated that most of the predicted target genes for these six miRNAs were related to neuronal apoptosis. We then investigated the dynamic changes of these six miRNAs atdifferent time-point (4hours,24hours,1week and3weeks) after SE.4、Meanwhile, neuronal survival and apoptosis in the hippocampus after SE wereevaluated by Nissl staining and TUNEL（Terminal deoxynucleotidyl transferase dUTP nickend labeling assay）.Results1、 After analyzing the initial results of deep sequencing, six deferentially expressed miRNA(miR-874-3p, miR-20a-5p, miR-345-3p, miR-365-5p, miR-764-3p and miR-99b-3p) werescreened out based on the criteria mentioned in Materials and Methods.2、 The expression of miR-874-3p, miR-20a-5p, miR-345-3p, miR-365-5p, miR-764-3p wassignificantly increased after SE (P <0.05), whereas the levels of miR-99b-3p was markedlydecreased (P <0.05). The results of qRT-PCR were in agreement with results from miRNAsequencing.3、 In the post-SE rat hippocampus, neuronal survival decreased, whereas neuronal deathincreased. Subsequent KEGG pathway analysis found that most of the predicted target geneswere associated with neuronal apoptosis, This observation suggested that these six miRNAsmay modulate neuronal apoptosis after SE.4、 Further analysis revealed that the levels of miR-365-5p and miR-99b-3p weresignificantly correlated with neuronal apoptosis after SE.ConclusionTaken together, our data suggest that SE induced by amygdala stimulation led toprofound changes in the miRNA expression profile in hippocampus. Meanwhile, miRNAs areimportant modulators of SE-induced neuronal apoptosis. These findings also open newavenues for future studies aimed at developing strategies against neuronal apoptosis after SE.