脑缺血后lncRNA调控神经元生存的机制研究

The mechanism of neuronal death after cerebral ischemia is not clear yet.Long non-coding RNA (lncRNAs) may epigenetically regulate the expression of target gene. Previous study with lncRNA microarray found lots of up-,or down-expressed lncRNAs in rat brain cortex 24 hours after intraluminal middle cerebral occlusion model.However, the mRNA levels of protein with nerve regenerative function were unchanged. Here, the cDNAs coding for lncRNAs,which overlap introns with protein coding mRNAs of nerve regenerative function, will be cloned by 5'- and 3'-RACE.The locus of both lncRNAs and target mRNA in the genome will be mapped. The distributions of lncRNAs in tissues and cells will be examined by qRT-PCR. The regulatory principles of lncRNAs on the transcription and translation of target gene after cerebral ischemia will be investigated by the protein immunoprecipitation and RNA pull-down methods. The functional domains in the lncRNAs will be validated. The neuroprotective potentials of knockdown of lncRNAs by siRNAs will be performed in cells and animals.This study can enrich the biological function of lncRNAs, and understand the mechanism of neuronal death after cerebral ischemia in the lncRNAs level,and may provide new targets for the treatment of ischemic stroke.

脑缺血性后神经元死亡的机制还不明确。长链非编码RNA（lncRNAs）可在表观遗传学水平调控靶基因的表达和功能。本课题前期用芯片检测发现大鼠局部脑缺血24小时后大脑皮层有大量lncRNA的异常表达，但具神经再生功能的蛋白编码mRNA水平未发生变化。本课题拟用RACE方法克隆表达水平上调、且与编码神经再生功能蛋白mRNA有内含子同源的lncRNAs; 分析编码lncRNAs与靶mRNA的基因组关联；用qRT-PCR等研究lncRNAs在组织细胞内的分布。利用蛋白质免疫共沉淀及RNA pull-down方法研究lncRNA对靶基因转录与翻译的调控机制；确定lncRNAs的功能结构域；通过RNA干扰（RNAi）沉默lncRNA表达，在体内外研究是否可逆转脑缺血后神经元的死亡。本研究可丰富lncRNAs的生物学功能，在lncRNAs水平了解脑缺血后神经元死亡的机制， 为治疗缺血性脑卒中提供新靶点。

缺血性脑卒中的神经元损伤机制尚不明确。长链非编码RNA（lncRNA）可在表观遗传学水平调控靶基因的表达和功能。课题前期用芯片技术得到脑缺血损伤大鼠缺血半影区表达异常的lncRNAs谱，本课题在该lncRNAs谱中筛选得到具有神经保护功能的lncRNA-N1LR。N1LR在小鼠脑缺血损伤体内外模型中的表达模式为：轻度损伤时表达上调，而中重度损伤则表达下调。利用RNAi技术和过表达重组腺病毒载体，构建N1LR低表达和过表达模型，进行体内外的功能研究：N2a细胞氧糖剥离/再灌注模型（OGD/R）中，N1LR加速细胞周期进程，促进细胞增殖，抑制细胞凋亡；在C57小鼠中动脉栓塞模型（MCAO）中，N1LR降低缺血梗死体积，抑制缺血半影区皮层神经元的凋亡，并减少缺血半影区皮层神经元的丢失。初步的机制研究表明:N1LR可能通过抑制p53蛋白Ser15磷酸化，抑制p53蛋白活化，从而发挥神经保护功能。本课题研究成果丰富了lncRNAs的生物学功能，在lncRNAs水平认识脑缺血后神经元死亡的机制，为缺血性脑卒中的治疗提供新思路。