长链非编码RNA MCL1在癫痫脑组织细胞焦亡中的作用及机制研究

Epilepsy is a kind of disease characterized by excessive synchronous discharge of neurons in the brain. The existing antiepileptic drugs are not effective, so it is urgent to find new therapeutic targets from a new perspective. Pyrosis is a newly discovered and confirmed programmed cell inflammatory death involved in the process of epilepsy. Experts in the same field and the applicant have found that long non-coding RNA (lncRNA) plays an important role in the process of epilepsy. Based on the preliminary basis, this study confirmed abnormal activation of pyrosis in brain tissues during the occurrence of epilepsy, and screening and informatics analysis of differentially expressed lncRNA in brain tissues of patients with temporal lobe epilepsy revealed a new, conservative lncRNA MCL1 with significantly decreased expression in the epileptic cortex and a target gene NLRC3/CARD8 related to pyrosis. Therefore, we hypothesized that lncRNA MCL1 regulates the pyrosis of hippocampal neurons in temporal lobe epilepsy. In this study, the applicant intends to further clarify the role and mechanism of lncRNA MCL1 in the pyrosis of hippocampal neurons in temporal lobe epilepsy by using RNA interference/overexpression, RIP, luciferase reporter gene and other technologies from clinical tissue, animal model and cell level, to provide theoretical basis and intervention targets for the pathogenesis and treatment of epilepsy.

癫痫是一种以脑部神经元过度同步化放电为特征的疾病，现有的抗癫痫药物疗效不佳，迫切需要从新的角度寻找治疗靶点。细胞焦亡是近年来发现并证实的一种新的参与癫痫发生过程的程序性细胞炎性死亡。同领域学者及申请人发现长链非编码RNA（lncRNA）在癫痫发生过程中发挥着重要作用。基于前期基础，本课题证实癫痫发生过程中脑组织细胞焦亡异常激活，对颞叶癫痫患者脑组织差异表达lncRNA进行筛选及信息学分析，发现了一个全新的、保守的、在癫痫皮层表达显著降低的lncRNA MCL1及与细胞焦亡相关的靶基因NLRC3/CARD8。因此，我们假设：lncRNA MCL1调控颞叶癫痫海马神经元焦亡。在本课题中，申请人拟从临床组织、动物模型及细胞水平，采用RNA干扰/过表达、RIP、荧光素酶报告基因等技术深入阐明lncRNA MCL1在颞叶癫痫海马神经元焦亡中的作用及机制，为癫痫发病机制及治疗提供理论基础和干预靶点。

癫痫是一种以脑部神经元过度同步化放电为特征的疾病，现有的抗癫痫药物疗效不佳，迫切需要从新的角度寻找治疗靶点。细胞焦亡是近年来发现并证实的一种新的参与癫痫发生过程的程序性细胞炎性死亡。同领域学者及申请人发现长链非编码RNA（lncRNA）在癫痫发生过程中发挥着重要作用。基于前期基础，本课题证实癫痫发生过程中脑组织细胞焦亡异常激活，对颞叶癫痫患者脑组织差异表达lncRNA进行筛选及信息学分析，发现了一个全新的、保守的lncRNA MCL1。qPCR及原位杂交检测显示lncRNA MCL1在癫痫状态下表达降低，且主要表达于神经元的细胞浆内。离体水平，采用慢病毒过表达载体过表达lncRNA MCL1并进行高通量测序和生物信息学分析，结果提示lncRNA MCL1与神经元的炎症反应密切相关。过表达lncRNA MCL1可抑制海人酸诱导的HT22海马细胞系细胞焦亡及炎症反应，而采用siRNA干扰lncRNA MCL1可加重上述反应。在体水平，采用腺相关过表达载体过表达lncRNA MCL1可抑制颞叶癫痫小鼠海马细胞焦亡和海马神经元缺失，并改善颞叶癫痫小鼠的自发性癫痫发作和戊四唑诱导的癫痫发作。采用RNA pulldown实验获得了277个可能与lncRNA MCL1直接相互作用的蛋白分子。此外，本研究还发现lncRNA MCL1在癫痫中的表达下调受炎症反应及炎症因子的调节。本课题发现了一个与癫痫发生密切相关的lncRNA分子，为癫痫发病机制及治疗提供理论基础和干预靶点。