摘要
为了验证miRNA-2127与p53基因的靶向关系,利用生物信息学软件预测p53与miRNA-2127的结合位点,全基因合成法合成该结合位点的野生型与突变型模板,并将其克隆到pmiR-RB-REPORT^(TM)双荧光素酶报告载体中,构建野生型与突变型重组双荧光素酶报告质粒。将293T细胞分为4组,分别共转染野生型报告质粒+阴性对照(NC)、野生型报告质粒+miRNA-2127、突变型报告质粒+NC、突变型报告质粒+miRNA-2127。检测各组细胞中荧光素酶活性差异,结果显示:共转染了野生型报告质粒+miRNA-2127的293T细胞与共转染了野生型报告质粒+NC组相比,荧光素酶活性显著降低(P<0.05);且突变型报告质粒+miRNA-2127组的相对荧光素酶活性显著高于野生型报告质粒+miRNA-2127(P<0.05),说明miRNA-2127能够靶向调控p53基因,且结合位点位于3′UTR区369—375间。
To verify the targeted relationship between miRNA-2127 and p53,the binding sites of p53 and miRNAs-2127 were predicted by bioinformatics software.The wild-type and mutant templates of the binding sites were synthesized by the whole gene synthesis method,and were cloned into pmiR-RB-REPORT^TM dual-luciferase reporter vector to construct the wild-type and mutant-type recombinant dual-luciferase reporter plasmids.The cultured monolayer 293T cells were divided into 4 groups including co-transfected wild-type reporter plasmid+NC control,co-transfected wild-type reporter plasmid+miRNA-2127,co-transfected mutant reporter plasmid+NC control and co-transfected mutant reporter plasmid+miRNA-2127,respectively.The difference of luciferase activity in each group was detected.The results showed that compared with co-transfected wild-type plasmid+NC control,the relative luciferase activity of 293T cells in the group with co-transfected wild-type plasmid+miRNA-2127 showed a significant decrease(P<0.05),and that of co-transfected mutant reporter plasmid+miRNA-2127 group was significantly higher than co-transfected wild-type reporter plasmid+miRNA-2127 group.The results indicated that miRNA-2127 could targeted regulate p53 and the binding sites were located between 369 and 375 in the 3′UTR region.
作者
张玉霞
袁小远
王友令
孟凯
Zhang Yuxia;Yuan Xiaoyuan;Wang Youling;Meng Kai(Institute of Poultry Sciences,Shandong Academy of Agricultural Sciences,Jinan 250023,China)
出处
《山东农业科学》
2019年第1期14-17,共4页
Shandong Agricultural Sciences
基金
山东省自然科学基金项目(ZR2017BC039)
山东省农业科学院高层次人才及创新团队引进计划项目
山东省农业科学院青年科研基金项目(2015YQN61)
