microRNA调控HIF-1α介导颞叶癫痫耐药的机制研究

The drug-resistance mechanism of temporal lobe epilepsy has become a big problem in the field of neuroscience which need to be overcome urgently. Supported by National Natural Science Fund of China (30900471), we have confirmed that HIF-1α is related to the drug-resistance mechanism of temporal lobe epilepsy. But the expression of HIF-1α is also regulated by microRNA, microRNAs is an important, newly discovered gene expression negative regulatory factors, which can ensure the timing and stability of gene expression. Based on above research and theory basis, this project aims to established the rat model of temporal lobe epilepsy kindled by coriaria lactone as the research subject, using microRNA array, Real-time fluorescent quantitative PCR and Western-blot method to test the changes of microRNA profile and HIF-1α expression in the process of model establishment, combined with the bioinformatics method，screen out the candidate microRNA which may negatively regulate the expression of HIF-1α.Then using many other methods, including transfection of the candidate microRNA and microRNA inhibitors to the lateral ventricle of epilepsy model, construction of luciferase reporter vector method and so on to verify the regulation of candidate microRNA on HIF-1α, reveal the effection on drug-resistance mediated by regulatory effects of microRNA on HIF-1α in temporal lobe epilepsy, in order to open a new way to study the drug-resistance mechanism of temporal lobe epilepsy.

颞叶癫痫的耐药机制是神经科学界急需攻克的难题。受国家青年自然科学基金项目的资助，我们已证实低氧诱导因子(HIF-1α)参与颞叶癫痫的耐药机制。但HIF-1α表达还受微小RNA(microRNA)的调控，microRNA 是近年来新发现的重要的保证基因表达时序性和稳定性的基因表达负向调控因子。在上述研究及理论基础上，本项目拟以马桑内酯点燃的颞叶癫痫大鼠模型为研究对象，利用microRNA array及实时荧光定量PCR等方法检测模型点燃过程中microRNA表达谱和HIF-1α表达的动态变化，结合生物信息学方法筛选出可能负调控HIF-1α的候选microRNA，通过向模型侧脑室转染合成的候选microRNA或其抑制剂、构建荧光素酶报告载体等方法验证候选microRNA对HIF-1α的调控作用，揭示microRNA调控HIF-1α对癫痫耐药的影响，从而为颞叶癫痫耐药机制的研究开辟新途径。

颞叶癫痫的耐药机制是神经科学界急需攻克的难题。微小RNA (microRNA) 是近年来新发现的重要的保证基因表达时序性和稳定性的基因表达负向调控因子。在前期已证实低氧诱导因子(HIF-1α)参与颞叶癫痫的耐药机制的基础上，本项目进一步研究HIF-1α表达受microRNA的调控，我们以马桑内酯点燃的颞叶癫痫大鼠模型为研究对象，利用microRNA array及实时荧光定量PCR等方法检测模型点燃过程中microRNA表达谱和HIF-1a表达的动态变化，结合生物信息学方法筛选出可能负调控HIF-1α的候选microRNA，通过向模型侧脑室转染合成的候选microRNA或其抑制剂、构建荧光素酶报告载体等方法验证候选microRNA对HIF-1α的调控作用，揭示microRNA调控HIF-1α对癫痫耐药的影响。研究结果显示：与HIF-1α表达呈反向变化的microRNA有microRNA-153, microRNA-494等，本项目进一步对反向变化的microRNA进行验证，发现其中microRNA-153通过调控HIF-1α有影响癫痫耐药的作用，为药物难治性癫痫的干预提供了潜在的生物学靶点。