lncRNA-Malat1调节海马苔藓纤维出芽在癫痫发生中的作用及机制研究

Epileptogenesis refers to a period that begins after the occurrence of brain insult and ends at the time of the appearance of the first spontaneous seizure. During this latency period, aberrant sprouting of granule cell axons (mossy fibers) and subsequent progressive formation of new recurrent excitatory circuits occurred and contribute to spontaneous recurrent seizures. So far, the mechanisms of mossy fiber sprouting remain unclear. Long non-coding RNAs (lncRNAs) are a novel class of non-coding transcripts, which had been identified in various regions of central nervous system. In our previous study, differentially expressed lncRNAs during epileptogenesis were identified by lncRNA microarray, among which lncRNA-Malat1 expression was found to be markedly upregulated in different time point of the latency period. Further bioinformatics analysis suggested that lncRNA-Malat1 may play its regulation mechanism on target gene-erk by phosphorylating SR protein. Based on the possible role of lncRNA-Malat1 on neural outgrowth found in our previous study, We hypothesed that lncRNA-Malat1 might activate mossy fiber sprouting and epileptogenesis by regulating gene-erk. Using AAV-CRISPR/Cas9, CHIP, 7T-MRI and other techniques, this project will further explore the role of lncRNA-Malat1 on epileptogenesis and its underlying mechanism in order to advance the mechanism of epileptogenesis and provide a new way of reversing epileptogenesis in clinical.

“癫痫发生”是脑部损伤后经一定时间潜伏期，从无到有出现慢性癫痫发作的过程。海马苔藓纤维出芽是癫痫发生的重要病理改变，其具体机制并不清楚。lncRNA在神经系统广泛表达，预实验在颞叶癫痫小鼠模型点燃过程中检测海马lncRNA表达谱，发现lncRNA-Malat1在“癫痫发生”潜伏期持续高表达，到癫痫自发发作期则显著下降。生物信息学分析提示Malat1可能磷酸化RNA剪接蛋白（SR蛋白）从而调控erk基因，结合本课题组前期研究已证实的Malat1有调控神经元轴突生长的作用，我们提出假说：lncRNA-Malat1通过磷酸化SR蛋白调控erk基因，促发海马苔藓纤维出芽导致“癫痫发生”。本项目拟用AAV-CRISPR/Cas9技术激活和敲除lncRNA-Malat1，在细胞和动物水平深入研究lncRNA-Malat1在“癫痫发生”中的作用和机制，为“癫痫发生”的机制研究及癫痫预防开辟新方向。

癫痫是第二大慢性神经系统疾病，具有药物难治率高、易复发的特点，已被世界卫生组织列为重点防治的五大神经精神类疾病之一。因此，预防癫痫发生是癫痫领域急需攻克的重大问题。阻止“癫痫发生”的关键在于阐明其确切的发生机制。鉴于lncRNA在神经系统广泛表达和重要地位，本研究旨在阐明lncRNA在癫痫发生中的作用。首先建立化学点燃的慢性癫痫动物模型，再通过高通量测序进行lncRNA检测，分析找出差异表达lncRNA并用qPCR验证，最后利用在体和离体实验，探究差异表达lncRNA对苔藓纤维出芽及兴奋性突触环路形成的作用及机制。通过探究，我们成功绘制出癫痫模型中的差异表达lncRNA谱，发现lncRNA-Malat1变化显著；通过组织 RNA-FISH 技术检测，在海马神经元中发现lncRNA-Malat1的亚细胞定位在细胞核核散斑中（用 SC35 蛋白标记）；通过检测慢性癫痫模型中lncRNA的表达情况，发现在癫痫发生过程中差异表达的lncRNA参与调控ERK/MAPK通路，而该通路的激活与癫痫发生密切相关，提示了该lncRNA在癫痫发生中的重要性。本研究发现揭示了“癫痫发生”的具体分子机制，能够进一步推动癫痫研究进展，为临床上癫痫的预防及治疗提供新的科学依据和理论支持。