miR-181b-5p在胆绿素治疗脑缺血再灌注损伤中的靶基因预测及鉴定  

Prediction and identification for target genes of miR-181b-5p in the treatment of cerebral ischemia-reperfusion injury with biliverdin

作　　者：罗靖 彭丽佳 邵建林[1] 熊莉 李俊杰[1] Luo Jing;Peng Lijia;Shao Jianlin;Xiong Li;Li Junjie(Department of Anesthesiology,First Affiliated Hospital of Kunming Medical University)

机构地区：[1]昆明医科大学第一附属医院麻醉科,昆明650032

出　　处：《重庆医科大学学报》2021年第10期1248-1254,共7页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81760248、81960250);云南省应用基础研究重点资助项目(编号:2018FA042);云南省高层次人才培养资助项目(编号:D-201632);云南产业技术领军人才培养资助项目(编号:YLXL20170054);俞卫锋专家工作站资助项目(编号:2017IC067);云南省应用基础研究联合资助项目(编号:2017FE468(-034));云南省教育厅科学研究基金资助项目(编号:2019J1250);昆明医科大学研究生创新基金资助项目(编号:2019S030)。

摘　　要：目的:对Small RNA测序中筛选出的rno-miR-181b-5p进行靶基因预测、生物信息学分析及鉴定,以期为miR-181b-5p在胆绿素治疗大鼠脑缺血再灌注损伤中的生物学作用奠定基础。方法:分别用TargetScan、miRDB、miRwalk 3个数据库预测rno-miR-181b-5p的靶基因,取3个数据库的交集靶基因进行基因功能富集分析(gene ontology)及信号通路富集分析(KEGG pathway analysis)。采用双荧光素酶基因报告实验对靶基因进行验证,同时通过RT-QPCR和Western blot检测大鼠血管内皮细胞中过表达miR-181b-5p后内皮细胞特异性分子1(Esm1)的表达变化。结果:3个数据库交集的靶基因共有28个,这些交集靶基因主要富集于调节刺激反应(P=0.013)、调节细胞增长(P=0.014)、积极调控(P=0.048)等生物过程GO条目中;而细胞成分层面主要富集于细胞内膜结合细胞器(P=0.045)、黏附连接(P=0.049)等GO条目中;分子功能层面,靶基因主要富集于与mRNA5’-UTR结合(P=0.017)、受体结合(P=0.032)、蛋白质结合(P=0.046)等GO条目中。信号通路主要富集于突触小泡循环(P=0.024)、基础转录因子(P=0.040)、RIG-I样受体信号通路(P=0.047)等通路中。双荧光素酶基因报告实验结果揭示了miR-181b-5p可以靶向调控Esm1基因。另外,RT-QPCR和Western blot结果显示在大鼠血管内皮细胞中过表达miR-181b-5p可负向调控Esm1。结论:rno-miR-181b-5p可能通过靶向调控Esm1基因而在胆绿素治疗大鼠脑缺血再灌注损伤中发挥生物活性作用。Objective:To predict the target genes of rno-miR-181b-5p selected from Small RNA sequence and to perform bioinformatics analysis and identification for laying a foundation for the biological function of miR-181b-5p in the treatment of cerebral ischemia-reperfusion injury with biliverdin. Methods :The target genes of rno-miR-181b-5p were screened through TargetScan,miRDB and miRwalk databases respectively. Then,bioinformatic analysis of these intersection target genes of the three databases were performed by gene ontology analysis and KEGG pathway analysis. Subsequently,dual luciferase reporter gene assays were performed to identify the targets,and the expression changes of endothelial cell specific molecule-1(Ems1)after overexpression of miR-181b-5p in rat vascular endothelial cells were detected by RT-QPCR and Western blot. Results:There were 28 target genes intersected by the three databases,which were mainly located in such GO items of biological process as regulating stimulus response (P =0.013),regulating cell growth(P =0.014) and positive regulation(P =0.048). The cellular component of these genes principally were involved in such GO items as intracellular membrane-bounded organelles(P=0.045)and adherens junction(P=0.049). At the molecular function level,these target genes were significantly involved in such GO items as mRNA 5’-UTR binding(P=0.017),receptor binding(P=0.032),and protein binding(P=0.046). The KEGG pathway analysis demonstrated that these genes were mainly enriched in synaptic vesicle circulation(P =0.024),basal transcription factors pathway(P =0.040)and RIG-I-like receptor signaling pathway(P=0.047),etc. The results of dual luciferase reporter gene assays suggested rno-miR-181b-5p could target and regulate Esm1 genes. Moreover,RT-QPCR and Western blot results also showed that overexpression of miR-181b-5p can negatively regulate Esm1 in rat vascular endothelial cells. Conclusion:rno-miR-181b-5p may play an important biologically role in the treatment of cerebral ischemia-reperfusion injury

关 键 词：rno-miR-181b-5p 靶基因 生物信息学 内皮细胞特异性分子1 

分 类 号：R363[医药卫生—病理学]