长链非编码RNA-HOTAIRM1介导的关键ceRNA紊乱对IDH1突变型脑胶质瘤相关癫痫的分子调控机制研究

Glioma accounts for about 60% of primary intracranial malignant tumor. More than 50% glioma patients suffer from seizure. IDH1 mutation significantly correlates with seizure in glioma. However, the underlying mechanism is still unclear. Our previous studies demonstrated that miR-196b was involved in epileptogenesis via promoting the proliferation and metabolism of tumor cells. LncRNA-HOTAIRM1 was down-regulated after IDH1 mutation and negatively correlated with miR-196b.GC1 was identified as the target gene of miR-196b. Here, we propose a hypothesis that HOTAIRM1 may induce seizure attacks via mediating the ceRNA disorder by sponging miR-196b away from GC1 and glutamate metabolic abnormality in glioma cells. To test the hypothesis, we intend to carry out the following studies: techniques such as RNA-RIP, luciferase reporter assay will be conducted to analyze HOTAIRM1 as a molecular sponge of miR-196b in regulating the GC1 at post-transcription regulation level. Primary cultured glioma cells, animal models and electrophysiological measurements will be utilized to verify the epileptic effect mediated by the IDH1 induced regulatory network. The present study is expected to provide insight into the molecular diagnosis/typing, prognosis evaluation and target therapy of tumor-related epilepsy.

脑胶质瘤约占颅内原发恶性肿瘤的60%，超过50%的脑胶质瘤患者以癫痫起病，IDH1突变与癫痫密切相关，但具体机制不清。前期研究证实miR-196b主要通过影响肿瘤细胞的增殖与代谢功能发挥致癫作用，IDH1突变后lncRNA-HOTAIRM1表达下调，HOTAIRM1与miR-196b呈负调控关系，GC1是miR-196b的作用靶点。我们提出假说：HOTAIRM1/miR-196b海绵调控GC1表达，影响肿瘤微环境中谷氨酸的代谢，诱导癫痫发生。本研究拟运用荧光素酶报告、RIP等实验技术分析HOTAIRM1作为分子海绵吸附miR-196b发挥对GC1的转录后调控机制，以及通过体外细胞系功能实验、动物模型、神经电生理技术等手段，从分子、细胞、组织以及临床整体水平探讨IDH突变后调控通路在胶质瘤相关癫痫发生发展中的作用。研究成果有助于对肿瘤性癫痫进行分子诊断与分型、预后评估及靶向药物研发。

脑胶质瘤约占颅内原发恶性肿瘤的60%，超过50%的脑胶质瘤患者以癫痫起病，IDH1突变与癫痫密切相关，但具体机制不清。前期研究证实miR-196b主要通过影响肿瘤细胞的增殖与代谢功能发挥致癫作用，IDH1突变后lncRNA-HOTAIRM1表达下调，HOTAIRM1与miR-196b呈负调控关系，GC1是miR-196b的作用靶点。本项目首先通过对HOTAIRM1的靶基因network分析，预测了上下游关键的分子靶点，GO、Pathway分析显示HOTAIRM1调控的分子网络大部分参与了细胞周期调控和细胞增殖过程；然后，我们通过HOTAIRM1基因RNA干扰和过表达慢病毒构建和转染技术，开展了HOTAIRM1及其关键靶基因在体外细胞系模型中的生物学功能研究，结果证实靶基因网络主要参与细胞周期调控，HOTAIRM1类似“脚手架”与EZH2（PRC2复合物的重要组成部分）结合，代表分子为细胞周期相关蛋白p16，p21，CyclinD1，CyclinE；接着，通过筛选U87稳转细胞系中HOTAIRM1高表达后的神经递质和炎症因子表达，发现以谷氨酸和组胺为代表的分子尤为突出；然后，我们运用蛋白质谱的检测手段，利用GO富集寻找到与谷氨酸、组胺密切相关的蛋白：EGFR、APP、CDK-5、PSEN1、STXBP1等，再通过荧光素酶报告试验的回馈验证，证实了除miR-196b之外的协调miRNA的共表达，发现miR-93-5p，miR-142-5p，miR-323a-5p，miR-365a-3p在HOTAIRM1高表达组的下调结果较为确切；最后通过临床验证，构建了脑胶质瘤相关癫痫的临床预测模型，此模型能够较为准确的预测患者的癫痫预后。综上所述，本研究基础结合临床，验证了脑胶质瘤相关癫痫中“LncRNA-MiRNA-蛋白”轴的关键分子通路，通过数据库挖掘和RNA测序分析，构建了脑胶质瘤相关癫痫的预测模型，并证实了它的临床预测价值，研究成果有助于对肿瘤性癫痫进行分子诊断、分子分型、预后评估及靶向药物研发。