miR-23b、miR-29b作为缺血性脑卒中分子标志物的临床和作用机制研究

The monitoring and assessment of ischemic stroke (IS) still need available and effective biomarkers. In our previous study, we found that serum miR-23b and miR-29b levels were significantly increased in IS patients and closely associated with the severity and onset time of IS. Bioinformatics predicted that miR-23b and miR-29b may regulate the expression of PTEN and FOXO3, involving in the ischemia-hypoxia induced apoptosis in neuronal cells. Consequently, we speculated that miR-23b and miR-29b may function as the potential biomarkers and participate in the development of IS through PTEN-AKT-FOXO3 pathway. To verify this hypothesis, we performed clinical, cellular and animal studies using plenty of biomedical methods, such as TaqMan Low Density Arrays, real-time quantitative PCR, luciferase reporter system, western blot, lentiviral transfection, et al. This project aimed to definite the clinical application value of miR-23b and miR-29b as the novel biomarkers for IS, ascertain their molecular mechanism involved in pathogenesis and progression of IS through PTEN-AKT-FOXO3 pathway, and provide a novel approach and theoretical basis for monitoring and assessment of IS.

缺血性脑卒中(IS)的病情监测与评估尚缺乏理想的生物标志物。本课题组前期研究发现IS患者血清miR-23b、miR-29b水平显著升高，且与疾病严重程度、发病时间有关；生物信息学分析显示miR-23b、miR-29b可能通过调控PTEN、FOXO3的表达参与缺血缺氧诱导的神经元凋亡过程。据此提出假设：血清miR-23b、miR-29b是IS潜在的分子标志物；可能通过调节PTEN-AKT-FOXO3信号通路参与IS的发生、发展。为证实该假设，拟从临床、细胞和动物实验研究，采用低密度芯片、实时荧光定量PCR、荧光素酶报告基因实验、Western blot、慢病毒载体转染等手段，明确miR-23b、miR-29b作为IS新型标志物的临床应用价值，探明其在PTEN-AKT-FOXO3通路中的靶向关系及参与IS发生、发展的分子作用机制，以期为IS的病情监测与评估提供新的途径和理论依据。

缺血性脑卒中(IS)的病情监测与预后评估尚缺乏理想的生物标志物。本项目通过低密度芯片分析结合实时荧光定量聚合酶链反应(qRT-PCR)技术，筛选并鉴定了IS患者的血清特异miRNAs表达谱，包括miR-23b-3p(miR-23b)、miR-29b-3p(miR-29b)、miR-181a-5p和miR-21-5p。通过采用单因素方差分析、Spearman相关性分析、受试者工作特征曲线分析、多因素Logistic回归分析等统计学方法和长期的跟踪随访，明确了上述血清特异miRNAs作为IS病情监测与预后评估分子标志物的临床应用价值。通过生物信息学分析，预测miR-23b-3p和miR-29b-3p的可能靶基因，构建了miR-23b-3p和miR-29b-3p可能参与的分子调控网络；进一步体外细胞实验，采用qRT-PCR、Western blot、荧光素酶报告基因实验、细胞凋亡检测及慢病毒感染等方法与手段，明确了miR-23b-3p和miR-29b-3p通过共同靶向基因磷酸酶张力蛋白同源物(PTEN)，miR-29b-3p亦可通过靶向叉头转录因子3(FOXO3)，调控PTEN-AKT-FOXO3信号通路，参与氧糖剥离诱导神经元凋亡过程的分子作用机制；并通过体内动物实验，进一步验证了miR-23b-3p和miR-29b-3p与PTEN-AKT-FOXO3信号通路间的靶向关系。本项目研究结果与结论为临床IS的病情监测与预后评估提供了新的途径和理论依据。本项目累计已发表论文8篇，其中科学引文索引(SCI)收录3篇，包括影响因子大于5分的SCI论文1篇；并多次参加国内学术交流荣获优秀论文奖励。培养博士研究生1名。项目投入经费24万元，支出22.864万元，各项支出基本与预算相符；剩余经费1.136万元，计划用于本项目研究后续支出。