miR-1290与RASAL2基因靶向关系的探索  

Exploration of the targeting relationship between miR-1290 and RASAL2 gene

作　　者：谭芳[1] 胡娟 李擎 杨晓燕[1] 雷小勇[1] AN Fang;HU Juan;LI Qing;YANG Xiaoyan;LEI Xiaoyong(Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study&Institute of Pharmacy and Pharmacology,University of South China,Hengyang,Hunan 421001,China)

机构地区：[1]南华大学湖南省分子靶标新药研究协同创新中心南华大学药物药理研究所,湖南省衡阳市421001

出　　处：《中南医学科学杂志》2021年第1期46-50,共5页Medical Science Journal of Central South China

基　　金：国家自然科学基金(81372579);湖南省自然科学基金(2019JJ50500,2017JJ2226);湖南省教育厅科学研究项目(17C1402);南华大学“大学生研究性学习和创新性实验计划”项目(2017XJYZ040,2018XJXZ348)。

摘　　要：目的通过构建RASAL23′-非翻译区(3′-UTR)双荧光素酶报告载体,探索RASAL2与非编码核糖核酸(ncRNA)miR-1290的靶向调控情况。方法借助在线数据库Targetscan预测miR-1290与RASAL2结合位点;利用聚合酶链反应(PCR)技术扩增RASAL2基因3′-UTR序列,并以GV272为载体将其连接,分别构建野生型GV272-RASAL2-wt 3′-UTR和突变型GV272-RASAL2-mut 3′-UTR荧光素酶报告基因重组质粒;并将miR-1290及相应阴性对照分别与这两种重组质粒共转染到293T细胞中,通过检测各组荧光素酶活性,探索miR-1290与RASAL2基因的结合情况。结果酶切和测序数据表明:野生型GV272-RASAL2-wt 3′-UTR及突变型GV272-RASAL2-mut 3′-UTR双荧光素酶报告载体构建成功;相较于miR-1290与突变型GV272-RASAL2-mut 3′-UTR共转染组,miR-1290与野生型GV272-RASAL2-wt 3′-UTR共转染组的荧光素酶活性无明显变化(P>0.05)。结论成功构建了RASAL23′-UTR的双荧光素酶报告基因载体,但miR-1290不能与RASAL2结合,不能抑制其表达。Aim To construct RASAL23′-untranslated region(3′-UTR)dual luciferase reporter vector,and explore the targeted relationship between non-coding ribonucleic acid(ncRNA)miR-1290 and RASAL2.Methods Online database Targetscan was used to predict the binding sites between miR-1290 and RASAL2.Polymerase Chain Reaction(PCR)was used to amplify RASAL23′-UTR sequence,which was then cloned into GV272 vector to construct wild type GV272-RASAL2-wt 3′-UTR and mutant GV272-RASAL2-mut 3′-UTR dual luciferase report plasmid.The miR-1290 or negative control was co-transfected into 293T cells with wild-type GV272-RASAL2-wt 3′-UTR or mutant GV272-RASAL2-mut 3′-UTR dual luciferase reporter plasmid,respectively.Cotransfection of miR-1290 or negative control with wild type GV272-RASAL2-wt 3′-UTR or mutant GV272-RASAL2-mut 3′-UTR dual luciferase report plasmid to 293T cells was conducted,and the luciferase activity was detected via dual luciferase reporter system to explore the combine relationship between miR-1290 and RASAL2 gene.Results The enzyme digestion and sequencing data showed that the wild type GV272-RASAL2-wt 3′-UTR and mutant GV272-RASAL2-mut 3′-UTR dual luciferase report vector were constructed successfully.Compared with the miR-1290 co-transfected with mutant GV272-RASAL2-mut 3′-UTR,the luciferase activity of miR-1290 co-transfected with wild-type GV272-RASAL2-wt 3′-UTR did not change significantly.Conclusion The RASAL23′-UTR dual luciferase reporter vector was constructed successfully.miR-1290 could not combine with RASAL23′-UTR,and inhibit its expression.

关 键 词：RASAL2 3′-非翻译区 双荧光素酶报告基因 miR-1290 

分 类 号：R730[医药卫生—肿瘤]