单纯疱疹性角膜炎免疫调节因子IL-12A与microRNA-21靶向作用关系的实验研究  

A preliminary study on the targeting effect relationship between herpes simplex keratitis immune regulatory factor IL-12A and microRNA-21

作　　者：曹楠珏 夏丽坤[1] 强薇[1] 张小娟[1] 张帆[1] 刘鹤南[1] 

机构地区：[1]中国医科大学附属盛京医院眼科,沈阳110004

出　　处：《中华眼科医学杂志（电子版）》2016年第5期219-226,共8页Chinese Journal of Ophthalmologic Medicine(Electronic Edition)

基　　金：国家自然科学基金(30772394);沈阳市科学技术计划项目(F13-318-1-03);沈阳市科学技术计划项目(F16-205-1-43)

摘　　要：目的寻找一种可同时调控白细胞介素-12A(IL-12A)等多个单纯疱疹性角膜炎(HSK)免疫调节因子的microRNA(miRNA)。鉴定miRNA与IL-12A等免疫调节因子的靶向调控作用并预测其在HSK中可能的作用机制。方法选取293T人胚肾细胞等培养材料进行实验。实验主要包括运用Target Scan v7.1、miRanda v2010、miRBase v2014及miRTar Base v6.0数据库进行多个免疫调节因子的miRNA预测,寻找共同的miRNA;运用miRBase数据库对各物种miR-21序列进行对比;通过RGator v3.0数据库分析hsa-miR-21在各组织中的表达情况;通过数据库检索,找出尚未验证的hsa-miR-21候选靶基因进行结合位点验证;通过构建IL-12A的3'-非翻译区(UTR)野生型及突变型载体质粒,将相应的质粒与hsa-miR-21模拟物转染到293T细胞中;通过双荧光素酶报告系统检测萤光素酶活性,初步验证两者的靶向结合作用;运用以上数据库进行hsa-miR-21的候选靶基因预测,得出候选靶基因集合;对候选靶基因集合进行基因功能富集分析和信号转导通路富集分析,并预测hsa-miR-21在HSK中可能的作用机制。结果通过以上数据库检索,发现hsa-miR-21是可以同时调控以上多个HSK免疫调节因子的miRNA,且各物种miR-21的序列基本一致。研究发现IL-12A是尚未验证的候选靶基因。双荧光素酶报告系统分析表明hsa-miR-21能够靶向作用于IL-12A的3'-UTR(t=-24.43,P<0.05),初步证明两者有靶向结合作用。结合数据库筛选出hsa-miR-21的候选靶基因共609个,其基因功能主要富集于调节细胞、细胞蛋白质代谢等过程(P<0.05),并涉及到丝裂原活化蛋白激酶(MAPK)信号转导通路、单纯疱疹感染通路、肿瘤坏死因子(TNF)信号通路、NF-κB信号通路及多种癌症通路(P<0.05)。研究结果表明,hsa-miR-21可能通过MAPK信号转导通路中的27个靶基因、单纯疱疹感染通路中的19个靶基因、TNF信号通路中的15个靶基因及NF-κB信号通路中的10个靶�Objective To find a microRNA （miRNA） which can simultaneously regulate IL-12A and other herpes simplex keratitis （HSK） immune regulatory factors. To explore its targeted regulation to IL-12A and other immune regulatory factors, and predict its possible mechanism in HSK. Methods 293T human embryonic kidney cells and other materials were selected for the experiment. The experiment mainly includes using TargetSean v7.1, miranda v2010, miRBase v2014 and miRTarBase v 6.0 database, the study attempted to find a common rniRNA through predicting the miRNA on multiple immune regulatory factors; it compared the miR-21 sequence of different species by using miRBase database ; it analyzed the expression of hsa-miR-21 in different tissues by using RGator v3.0 database. By searching the above databases, the study found an unverified hsa-miR-21 candidate target gene to verify binding site. By constructing wild type and mutant type plasmid of IL-12A＇s 3＇-UTR, hsa-miR-21 mimics and corresponding plasmid were transferred into 293T cells. The luciferase activity was tested by using luciferase reporter system, and targeted binding effect of hsa-miR-21 and IL-12A＇s 3＇-UTR was preliminary verified. To predict target genes of hsa-miR-21 by using the above databases, a collection of candidate target genes was derived; Gene Ontology analysis and KEGG pathway enrichment analysis were preformed on prediction results to predict and find out its possible mechanism in HSK. Results Through the above databases analysis, hsa-miR-21 can simultaneously regulate and control more than one of HSK immune regulatory factors. The sequences of the species of miR-21 were basically identical. It was found that IL-12A is the unverified candidate target gene and dual luciferase reporter system analysis showed that miR-21 can be targeted to IL-12A＇s 3＇-UTR （t =-24.43,P〈0.05）, thus their targeted binding effect was preliminarily proved. To screen out 609 candidate target genes, and the gene functions were enriched in regulation of cel

关 键 词：MICRORNA-21 白细胞介素-12A 单纯疱疹性角膜炎 生物信息学 双荧光素酶报告系统 

分 类 号：R772.21[医药卫生—眼科]