构建ECE1 3'UTR荧光素酶报告基因载体验证其与miR-199a-5p靶向关系

Construction of ECE1 3'UTR Dual Luciferase Reporter Vector and Verification of the Targeted Relationship with miR-199a-5p

目的:通过构建双荧光素酶报告基因重组质粒验证miRNA-199a-5p(miR-199a-5p)与ECE1基因的靶向调控关系。方法:通过microRNA靶基因预测软件Targetscan获取miR-199a-5p与ECE1基因3'UTR潜在的互补结合位点,PCR技术扩增ECE1基因3'端非翻译区,将此序列与miR-199a-5p mimics或空质粒(NC)共转染到pmirGLO-ECE1-野生型(WT)的293细胞里;将此序列突变序列与miR-199a-5p mimics或NC共转染到pmirGLO-ECE1-突变型(MUT)的293细胞里,共四组,检测四组细胞中荧光素酶活性。结果:成功构建双荧光素酶报告基因重组质粒pmirGLO-ECE1-WT和pmirGLO-ECE1-MUT。与野生型NC(4.30±0.53)组及突变型miR-199a-5p mimics组(4.465±0.3968)比较,野生型miR-199a-5p mimics组(1.686±0.4098)荧光素酶活性明显降低,P<0.05,有显著差异;突变型miR-199a-5p mimics组(4.465±0.3968)与突变型miR-199a-5p NC(4.18±0.498)组及野生型NC(4.30±0.53)组两两比较,P>0.05,无明显差异。结论:miR-199a-5p与野生型ECE1存在结合位点,miR-199a-5p对野生型ECE1重组质粒荧光活性有明显的抑制作用,证实miR-199a-5p能够靶向调控ECE1基因。

Objective: To identify the targeted-regulating relationship between miR-199 a-5 p and ECE1 via constructing luciferase reporter gene vector. Methods: The potential complementary binding sites of miR-199 a-5 p and ECE1 were predicted by Targetscan. The3’-untranslated regions(3’UTR) of ECE1 fragment amplified by PCR was cloned into luciferase reporter vector pmirGLO and constructed the recombinant plasmids pmirGLO-ECE1-WT and pmirGLO-ECE1-MUT, respectively, 293 T cells were cultured. Two recombinant plasmids were cotransfected into 293 T cells with miR-199 a-5 p group mimics or negative control, four groups totally. The luciferase activity was detected by dual luciferase reporter gene system. Results: The sequences of pmirGLO-ECE1-WT and pmirGLO-ECE1-MUT were cloned into pmirGLO successfully. Compared with the wild-type NC(4.30 ±0.53)group and the mutant miR-199 a-5 p mimics group(4.465±0.3968), the luciferase activity of the wild type miR-199 a-5 p mimics group(1.686±0.4098) was significantly reduced,P<0.05. The mutant miR-199 a-5 p mimics group(4.465±0.3968) was compared with the mutant miR-199 a-5 p NC(4.18±0.498) group and the wild-type NC(4.30±0.53), P>0.05, no significant difference, and concluded that miR-199 a-5 p had a significant inhibitory effect on wild-type ECE1 recombinant plasmid fluorescence activity, and confirmed that miR-199 a-5 p could target the ECE1 gene. Conclusion:The miR-199 a-5 p had binding sites with wild-type ECE1. The miR-199 a-5 p inhibited the luciferase activity of ECE1 wild type vector,which indicates ECE1 gene could be targeted regulated by miR-199 a-5 p.