长链非编码RNA对缺血性中风中经络不同证候的影响及功能机制研究

Disease burden of ischemic stroke (IS) is heavy, the pathogenesis of IS has not been elucidated. In the prevention and treatment of IS, it is meaningful to explore the differentiation basis and therapy target with different IS syndrome types. In our prophase research, through lncRNA microarray, qRT-PCR and cytoplasm-nucleus separation experiment, we found three lncRNAs located in nucleus and associated with the occurrence of IS. Therefore, hypothesis is as follows: these three lncRNAs may influence the occurrence and development of different traditional Chinese medicine (TCM) syndrome types with IS attacking on meridians. In order to verify the hypothesis, we firstly identify the diagnosis and prognosis lncRNA biomarkers of wind-phlegm blocking collaterals symptom and wind-yang disturbance syndrome with IS attacking on meridians. Secondly, we use CCK-8, flow cytometry and transwell assay in vivo and TTC staining, balance beam experiment and Zea Longa score in vitro to investigate the biological functions of lncRNAs. Finally, we screen and validate the RNA binding proteins of candidate lncRNA biomarkers through ChIRP-MS and RIP experiments. In this research, we can provide biological objective evidences for the differentiation with IS attacking on meridians, basic data for revealing the essential features of TCM syndromes, and clinical therapy targets for the prevention and treatment of IS.

缺血性中风（IS）疾病负担沉重，发病机制尚未阐明，探索IS辩证依据及治疗靶点对IS的防治具有重要意义。申请人前期运用lncRNA芯片、qRT-PCR、核质分离等技术鉴别出定位于细胞核中的3个lncRNAs与IS的发生相关。据此假设：3个lncRNAs可能影响IS中经络不同证候的发生发展。为验证假设，我们先从人群水平鉴别出IS中经络风痰瘀阻证、风阳上扰证的诊断及预后的lncRNAs生物标志物。然后，在细胞水平上运用CCK-8、流式细胞术、Transwell实验，在动物水平上通过TTC染色法、平衡木实验、Zea Longa评分，以探索lncRNA的生物学功能。再运用ChIRP-MS、RIP等技术，筛选及验证与候选lncRNAs结合的RNA结合蛋白。研究结果可为IS中经络辨证提供生物学客观依据，为阐明中风“证”本质提供基础数据，为IS防治提供治疗靶点。

缺血性中风（IS）疾病负担沉重，发病机制尚未阐明，探索IS辩证依据及治疗靶点对IS的防治具有重要意义。本项目首先通过大样本病例对照研究，从人群水平鉴别出可能与IS中经络风痰瘀阻证、风阳上扰证发生相关的lncRNA G066582和SERPINB9P1。然后，通过在A172细胞建立氧糖剥夺/再灌注（OGD/R）模型，运用细胞转染、qRT-PCR、CCK-8、流式细胞术、EdU细胞增殖实验、Transwell实验，发现lncRNA G066582可降低细胞活性、促进细胞凋亡、降低细胞迁移和增殖能力，而lncRNA SERPINB9P1可抑制细胞凋亡、增强细胞活力，从而阐明G066582和SERPINB9P1的生物学功能。再运用ChIRP-MS实验，筛选出可能与lncRNA SH3BP5-AS1特异性结合的7个蛋白（EL-S-270、LMNA、PS1TP5BP1、EEF1A1、PKM、KRT78、VIM），以及6个SERPINB9P1的潜在结合蛋白（MAP2K3、HNRNPK、HSPA2、VDAC1、PARK7、EEF1A1）。本项目鉴别出了与IS中经络风痰瘀阻证、风阳上扰证发生相关的lncRNAs，阐明了其生物学功能，筛选出与lncRNAs特异性结合的RNA结合蛋白。本项目研究成果可为IS中经络辨证提供生物学客观依据，为阐明中风“证”本质提供基础数据，为IS防治提供治疗靶点。本项目已发表3篇学术论文，3篇论文被期刊录用。培养了硕士研究生4名，博士研究生2名，均为在读研究生。还有2名课题组成员晋升职称。