构建ECE13'UTR荧光素酶报告基因载体验证其与miR-199a-5p靶向关系
Progress in Modern Biomedicine(2019)
Abstract
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-ECE 1-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-5pmimics组(4.465±0.3968)与突变型miR-199a-5pNC(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.
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