lincRNA-p21调控DNA甲基化介导颞叶内侧癫痫耐药的机制研究

The mesial temporal lobe epilepsy is the most common type of refractory epilepsy. The onset mechanism of drug-resistance remains unclear, which has raised great global awareness. The evidence has accumulated that abnormality in the expression and function of lncRNA plays a crucial role in human pathogenesis. Studies have shown that lincRNA-p21 promotes DNA methylation through binding with DNA methyl transferase-1 (DNMT1), engaging in epigenetic regulation of the target genes. We previously established kainic acid (KA) induced chronic epilepsy mouse model, and compared the difference in lncRNA expression in hippocampal region between AEDs-sensitive and AEDs-resistant mice using lncRNA microarray chip. The results indicate that down-regulation of lincRNA-p21 is closely associated with refractory epilepsy. We therefore hypothesize that down-regulation of lincRNA-p21 interferes with MDR-1 gene methylation by DNMT1, leading to elevated expression of MDR1/P-gp, and further engages in mechanism of refractory epilepsy.This project intends to construct lentivirus vectors to upregulate and silence the expression of lincRNA-p21 in animal and cell models, to explore the essential role of lincRNA-p21 in the onset of refractory epilepsy using molecular biological and behavioral measures, and to provide novel evidence and cornerstone for further therapies for refractory epilepsy targeting lncRNA.

颞叶内侧癫痫是临床最常见的耐药性癫痫类型，其耐药机制不清，已成为公认的科学问题和临床难点。研究表明lncRNA表达及功能异常是人类疾病产生的重要原因。lincRNA-p21招募DNA甲基转移酶DNMT1促进DNA甲基化修饰，参与靶基因表观遗传学调控。课题组前期通过海人酸小鼠慢性颞叶癫痫模型，采用表达谱芯片比较药物敏感组与耐药组海马组织lncRNA表达差异，发现lincRNA-p21表达下调与癫痫耐药性具有密切关系，但其具体作用机制不明。我们提出假说，lincRNA-p21下调降低DNMT1对多耐药基因MDR1甲基化修饰，致使MDR1/P-gp过度表达，进而导致癫痫耐药。本项目拟应用慢病毒载体过表达或沉默lincRNA-p21，在动物和细胞模型深入研究lincRNA-p21在癫痫耐药中的作用及分子机制，为以lncRNA为靶点的耐药癫痫治疗提供新的科学依据和理论基础。

颞叶内侧癫痫是临床最常见的耐药性癫痫类型，其耐药机制不清,已成为公认的科学问题和临床难点。研究表明lncRNA表达及功能异常是人类疾病产生的重要原因。lincRNA-p21招募DNA甲基转移酶DNMT1促进DNA甲基化修饰，参与靶基因表观遗传学调控。课题组前期通过海人酸小鼠慢性颞叶癫痫模型，采用表达谱芯片比较药物敏感组与耐药组海马组织lncRNA表达差异，发现lincRNA-p21表达下调与癫痫耐药性具有密切关系，但其具体作用机制不明。本研究通过构建癫痫动物模型及耐药性评估筛选，获取药物敏感组及耐药组小鼠海马组织，提取RNA行lncRNA表达谱芯片比较两组lncRNA表达差异，利用UCSC、GO及EMSENBL等相关软件分析后，鉴定与筛选对DNA甲基化可能有作用的关键lncRNA，结果提示lincRNA-p21可能是颞叶内侧癫痫耐药的关键lncRNA。利用甲基化特异性PCR检测MDR1启动子甲基化程度，利用Northern和Western检测MDR1表达水平。我们发现在耐药性颞叶内侧癫痫lincRNA-p21表达降低，而MDR1甲基化明显升高，提示MDR1甲基化水平可能与lincRNA-p21存在相关性。.进一步通过构建慢病毒载体过表达或沉默lincRNA-p21，采用行为学方法评估药物治疗反应性，结果提示在lincRNA-p21过表达的模型小鼠中，其慢性自发性癫痫对于卡马西平及丙戊酸钠药物的治疗反应性较lincRNA-p21沉默组良好，癫痫发作频率明显减少，其发作烈度较沉默组低，提示调控lincRNA-p21的确能够影响模型小鼠的癫痫耐药性。另一方面，我们通过进一步检测不同组别的MDR1甲基化水平，发现在lincRNA-p21过表达组，MDR1甲基化水平较沉默组降低，利用PCR及Western Blot检测P-gp蛋白水平，发现P-gp水平也相应降低。通过体外实验，发现在lincRNA-p21过表达组中对于罗丹明的转运效能较lincRNA-p21沉默组良好。进一步通过RIP和ChIP实验证实，lincRNA-p21对靶基因的调控是通过与DNMT1结合从而发挥其作用的。根据实验结果初步分析，我们认为lincRNA-p21水平改变的确引起MDR1甲基化及表达水平改变，为癫痫耐药性的治疗提供了可能的治疗靶标。