| 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 |
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| 引用本文： | 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].现代生物医学进展,2019,19(2):207-210. |
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| 作者姓名： | 鲍 宁 陈凤收 姜艳华 方 波 马 虹 |
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| 作者单位： | 中国医科大学附属第一医院麻醉科;沈阳市妇婴医院麻醉科 |
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| 基金项目： | 国家自然科学基金项目（81771342） |
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| 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,P0.05,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,P0.05,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。
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| 关 键 词： | MiRNA-199a-5p ECE1基因 荧光素酶报告基因 3''端非翻译区 |
| 收稿时间： | 2018/3/31 0:00:00 |
| 修稿时间： | 2018/4/27 0:00:00 |
Construction of ECE1 3'UTR Dual Luciferase Reporter Vector and Verification of the Targeted Relationship with miR-199a-5p |
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| BAO Ning,CHEN Feng-shou,JIANG Yan-hu,FANG Bo and MA Hong.Construction of ECE1 3''UTR Dual Luciferase Reporter Vector and Verification of the Targeted Relationship with miR-199a-5p[J].Progress in Modern Biomedicine,2019,19(2):207-210. |
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| Authors: | BAO Ning CHEN Feng-shou JIANG Yan-hu FANG Bo and MA Hong |
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| Institution: | 1 Department of Anaesthesiology ,The First Hospital of China Medical University Liaoning province, Shenyang Liaoning, 110001, China;2 Shenyang Women''s and Children''s Hospital, Shenyang Liaoning, 110003, China,Department of Anaesthesiology ,The First Hospital of China Medical University Liaoning province, Shenyang Liaoning, 110001, China;,Department of Anaesthesiology ,The First Hospital of China Medical University Liaoning province, Shenyang Liaoning, 110001, China;,Department of Anaesthesiology ,The First Hospital of China Medical University Liaoning province, Shenyang Liaoning, 110001, China; and Department of Anaesthesiology ,The First Hospital of China Medical University Liaoning province, Shenyang Liaoning, 110001, China; |
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| Abstract: | |
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| Keywords: | MiRNA-199a-5p ECE1 gene Luciferase reporter gene 3''UTR |
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