双荧光素酶报告系统鉴定miRNA-3133对200kD黏着斑激酶家族相互作用蛋白基因的靶向调控作用

To identify targeted regulation effect of miRNA-3133 on FIP200 gene by dual luciferase reporting system

目的通过构建分子量大小为200 kD的黏着斑激酶家族相互作用蛋白(FIP200) 3’UTR双荧光素酶报告基因载体,研究FIP200与mi R-3133的靶向关系。方法采用生物信息学软件targetscan预测mi R-3133与FIP200 3’端非翻译区(3’UTR)潜在的互补结合位点;利用PCR扩增FIP200基因3’UTR序列,并克隆到psicheck2载体中,构建FIP200野生型和突变型重组双荧光素酶报告基因载体;将野生型psicheck2-FIP200-wt 3’UTR或突变型psicheck2-FIP200-mut 3’UTR双荧光素酶报告质粒分别与mi RNA-3133 mimics或mimics-NC共转染CNE-1细胞;通过双荧光素酶报告系统检测各组荧光素酶活性,观察mi RNA-3133对FIP200表达的影响。结果生物信息学分析显示FIP200 3’UTR与mi RNA-3133有潜在的结合位点,酶切和测序结果表明野生型和突变型双荧光素酶报告载体构建成功;双荧光素酶报告系统检测显示,mi RNA-3133可抑制野生型FIP200载体的荧光素酶的表达(P <0.05),而对突变型载体的荧光素酶表达无影响(P> 0.05)。结论成功构建FIP200基因3’UTR的双荧光素酶报告载体,mi RNA-3133可以直接靶向调控FIP200基因的表达。

Objective Focal adhesion kinase family interacting protein of 200 kD( FIP200) 3 ’UTR dual luciferase reporter vector was constructed to study the targeting relationship between FIP200 and mi R-3133. Methods Bioinformatics software targets was used to predict the potential complementary binding sites between mi R-3133 and the 3 ’ untranslated region( 3’UTR) of FIP200;the 3’UTR sequence of FIP200 gene was amplified by PCR and cloned into psicheck2 vector to construct FIP200 wild-type and mutant recombinant double luciferase reporter gene vector;wild-type psicheck2-FIP200-wt 3’UTR or mutant psicheck2-FIP200-mut 3 ’UTR dual luciferase reporter plasmid were constructed and co-transfected with mi RNA-3133 mimics or mimics-NC to CNE-1 cells;the luciferase activity of each group was detected by the double luciferase reporting system,and the effect of mi RNA-3133 on FIP200 expression was observed. Results Bioinformatics analysis shows potential binding sites of FIP200 3’ UTR and mi RNA-3133,digestion and sequencing results indicate successful construction of wild-type and mutant dual-luciferase reporter vectors;the detection of dual luciferase reporter system showed that mi RNA-3133 could inhibit the expression of luciferase in wild-type FIP200 vector( P < 0. 05),but had no effect on the expression of luciferase in mutant vector( P > 0. 05). Conclusion The double luciferase reporter vector of 3’UTR of FIP200 gene was successfully constructed. mi RNA-3133 can directly target and regulate the expression of FIP200 gene.