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.
作者
谭芳
胡娟
李擎
杨晓燕
雷小勇
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)
出处
《中南医学科学杂志》
CAS
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)。
