| 鲍 宁,陈凤收,姜艳华,方 波,马 虹.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[J].,2019,19(2):207-210 |
| 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 |
| 投稿时间：2018-03-31 修订日期：2018-04-27 |
| DOI：10.13241/j.cnki.pmb.2019.02.002 |
| 中文关键词: MiRNA-199a-5p ECE1基因 荧光素酶报告基因 3'端非翻译区 |
| 英文关键词: MiRNA-199a-5p ECE1 gene Luciferase reporter gene 3'UTR |
| 基金项目:国家自然科学基金项目（81771342） |
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| 中文摘要: |
| 摘要 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。 |
| 英文摘要: |
| ABSTRACT Objective: To identify the targeted-regulating relationship between miR-199a-5p and ECE1 via constructing luciferase reporter gene vector. Methods: The potential complementary binding sites of miR-199a-5p and ECE1 were predicted by Targetscan. The 3'-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, 293T cells were cultured. Two recombinant plasmids were cotransfected into 293T cells with miR-199a-5p group mimics or negative control, four groups totally. The luciferase ac- tivity 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-199a-5p mimics group (4.465±0.3968), the luciferase activity of the wild type miR-199a-5p mimics group(1.686±0.4098) was significantly reduced, P<0.05. The mutant miR-199a-5p mimics group(4.465±0.3968) was compared with the mutant miR-199a-5p 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-199a-5p had a significant inhibitory effect on wild-type ECE1 recombinant plasmid fluorescence activity, and confirmed that miR-199a-5p could target the ECE1 gene. Conclusion: The miR-199a-5p had binding sites with wild-type ECE1. The miR-199a-5p inhibited the luciferase activity of ECE1 wild type vector, which indicates ECE1 gene could be targeted regulated by miR-199a-5p. |
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