Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies.微小RNA（MicroRNA，miRNA）海绵是一类带有重复miRNA反义序列的转录本，可从内源性靶点中隔离miRNA。miRNA海绵是用于miRNA功能丧失研究的宝贵工具，可应用于体外（in vitro）和体内（in vivo）实验。本研究开发了一种快速且灵活的miRNA海绵构建方法，并在多种检测实验中验证了其有效性。通过单次定向连接反应，我们可构建拥有10个及以上miRNA结合位点的海绵序列。荧光素酶（Luciferase）与AGO2免疫沉淀（immunoprecipitation，IP）实验证实，miRNA可与海绵序列有效结合。通过GFP竞争实验，我们发现：在miRNA结合位点中存在中心错配的miR-19海绵可作为高效的miRNA抑制剂，而带有完全互补反义结合位点的海绵则无此效果。对miRNA海绵表达水平的定量分析表明，这一现象至少部分源于完全互补反义海绵转录本的降解。最后，本研究证实，相较于单独抑制单个miRNA，联合抑制miR-17~92基因簇（miR-17~92 cluster）的多个miRNA可实现更显著的生长抑制效果。综上，本研究描述并验证了一种可快速构建用于miRNA功能丧失研究的miRNA海绵的方法。