circ-DROSHA通过ceRNA方式调控Ephrin-B3在癫痫突触重塑中的机制研究

Studies have suggested that circ-DROSHA expressed lower in the temporal lobe epilepsy patients, which play a critical role in the synaptic function maintaining. Our previous studies have showed the axon guidance moleculese ephrin-B3 decreased in epileptic rats. Therefore, we hypothesize circ-DROSHA can modulate Ephrin-B3 in the synaptic remodeling post epilepsy. In this study, we first investigate the expression of circ-DROSHA and ephrin-B3 gene by retrovirus transfection, and then techniques of qPCR, immunoprecipitation would be applied to investigate the role of circ-DROSHA and ephrin-B3 in synaptic remodeling. Furthermore, we detect the expression of ephrin-B3 after circ-DROSHA overexpression and knockdown to clarify the correlation of circ-DROSHA and ephrin-B3. we will clarify the mechanism of circ-DROSHA regulating ephrin-B3 by using biological information analysis and explored a group of miRNAs which might influence ephrin-B3 and circRNA immunoprecipitation （circRIP）was used to verify the miRNAs bind with circ-DROSHA. Then intervene the expression of miRNAs to investigate the influence in ephrin-B3 and find the way of circ-DROSHA regulate Ephrin-B3 by competing endogenous RNAs（ceRNA） in synaptic remodeling in epilepsy, which may provide therapeutic strategies for epilepsy.

研究表明环状circ-DROSHA在癫痫脑组织中表达下调，参与癫痫突触重塑的过程。前期我们研究显示轴突导向分子Ephrin-B3在匹罗卡品癫痫大鼠脑组织中有下调，因此我们提出科学假设circ-DROSHA靶向Ephrin-B3参与突触重塑。本研究拟采用逆转录病毒干预、qPCR、蛋白质共沉淀等技术探讨两者在癫痫大鼠海马神经元突触重塑中的作用；同时应用慢病毒过表达和knockdown circ-DROSHA后，检测Ephrin-B3表达变化，分析两者的正相关性；结合生物信息学的方法筛选与两者有共同结合位点的miRNAs，环状RNA免疫沉淀（circRIP）验证结合力强的miRNAs，通过干预其表达后检测Ephrin-B3表达变化，探索circ-DROSHA通过竞争性内源RNA（ceRNA）方式调控Ephrin-B3在癫痫突触重塑中的作用，为癫痫的治疗提供新的理论依据。

本研究拟研究环状circ-DROSHA及轴突导向蛋白Ephrin-B3在氯化锂-匹罗卡品致痫大鼠海马突触重塑中的作用，结合RNA测序、电镜三维成像和免疫荧光技术，探讨circ-DROSHA是否可以通过Ephrin-B3蛋白影响癫痫海马突触重塑的作用及机制。但在实验进程中，我们发现circ-DROSHA的本地表达较低，经反复验证，我们对研究内容进行了适当调整。调整后实验方案转向Reelin信号通路。通过研究Ephrin-B3与Reelin信号通路中Reelin蛋白和下游磷酸化Dab1（p-Dab1）蛋白在癫痫的表达变化，我们发现Ephrin-B3在癫痫中的表达下降；通过立体定位注射Ephrin-B3激动剂EphB3-Fc后，Reelin蛋白和p-Dab1蛋白表达增加，同时癫痫大鼠痫性发作的频率和持续时间减少，脑电图尖波和棘波发放频率减少，幅度下降，提示Ephrin-B3可能通过调控Reelin信号通路影响癫痫的发生发展。课题旨在为进一步揭示Ephrin-B3和Reelin信号通路在癫痫发生发展中的作用及其机制提供实验室依据。