LncRNA参与Arc调控海马神经元突触重塑在癫痫发生中的机制研究

Epilepsy is one of the most common and serious chronic nervous system diseases. Many studies have revealed that the expression of activity-regulated cytoskeletal (Arc) gene was induced by neuronal activity such as seizures, so that the Arc gene would quickly be expressed in hippocampal neuron dendrites and may be involved in the process of synaptic remodel, which may be one of the important mechanisms in the formation of aberrant networks of epilepsy. Many publications have shown that long non-coding RNAs (lncRNAs) may participate in the process of genetic transcription and expression and play an important role in neurological diseases. Our previous studies showed a high correlation between the expression of some lncRNA and Arc mRNA in hippocampus of epileptic rats induced by chloride lithium-pilocarpine, which suggested that lncRNA may regulate the process of Arc transcription. In this protocol, we will conduct a systematic study by using the lncRNA and mRNA chip, along with luciferase reporter gene system, immunoprecipitation tools and bioinformatics method to clarify the possible effects of Arc gene in hippocampal neuron synaptic remodel of epilepsy in rats. Furthermore, we will try to illuminate the underlying mechanism of how lncRNA affecting Arc gene expression in the process of epileptic hippocampal neuron synaptic remodeling. These findings will provide new theoretical and experimental basis for verifying mechanisms of epilepsy and also propose potential new therapeutic strategies for epileptic treatment.

癫痫是最常见且严重的慢性神经系统疾病。Arc基因在受到痫性发作等刺激后，迅速表达于海马神经元树突并参与其后突触重塑过程，是癫痫异常神经网络形成的重要机制之一。研究显示lncRNA(long non-coding RNA)可在多层面上调控基因转录，在神经系统疾病的发生发展中起重要作用。我们前期研究显示某些lncRNA与Arc mRNA在匹罗卡品致痫大鼠海马神经元中的表达具有高度相关性，提示lncRNA可能参与Arc表达调控。因此，本项目拟采取lncRNA芯片和mRNA芯片，联合生物信息学方法，荧光素酶报告基因系统、染色质免疫沉淀等手段进行系统研究，明确Arc在癫痫海马神经元突触重塑中的作用，并进一步探讨lncRNA参与Arc表达的调控机制及其对海马神经元突触重塑的影响，为癫痫的发生发展以及临床治疗提供理论基础和实验依据。

本项目采用基因芯片技术，研究了lncRNA、miRNA、mRNA在氯化锂-匹罗卡品致痫大鼠模型海马神经元中的表达改变，并用PCR实验验证了芯片结果。根据芯片结果构建了癫痫相关的lncRNA相关ceRNA调控网络，该网络的构建为癫痫lncRNA-miRNA异常调控网络机制提供了新思路。进一步探讨了lncRNA Bc-1和Kcna2-AS、miR-33和miR-211及其靶基因ARC在癫痫持续状态后各时点表达水平变化，通过双荧光素酶报告基因实验验证了lncRNA Bc-1和Kcna2-AS、miR-211和miR-33与Arc之间具有相互作用关系，最后通过过表达和RNA干扰实验验证了miR-33对Arc具有靶向调控作用。课题组已基本完成本项目研究计划，为lncRNA和miRNA参与癫痫的发病机制提供了新的思路。