miRNA expression in E18.5 murine total lung tissue

Knockout of the ubiquitously expressed microRNA-17~92 cluster in mice produces a lethal developmental lung defect. We validated the equally widely expressed pro-apoptotic Bim gene as joint target of miR-17~92 cluster members. To study the contribution of miR-17~92:Bim interaction to miR-17~92 overall function, we set up a system of conditional mutagenesis of the Bim 3' UTR. Blocking miR-17~92:Bim interaction early in development phenocopied the lethal lung phenotype of miR-17~92 ablation. Thus, despite hundreds of overall predicted targets vital miRNA functions can be mediated by a single target gene. Total RNA was extracted from 5 wild type and 4 Bim3'UTRmut/mut E18.5 total lung tissues. 100 ng of total RNA per sample was used as input for nCounter gene expression analysis (NanoString Technologies).在小鼠中全身性敲除普遍表达的微RNA-17~92簇（microRNA-17~92 cluster），可引发致命的发育性肺缺陷。本研究证实，同样广泛表达的促凋亡Bim基因（Bim gene）是微RNA-17~92簇各成员的共同靶基因。为探究微RNA-17~92与Bim的相互作用对该簇整体功能的贡献，我们构建了针对Bim基因3'非翻译区（3' untranslated region，3' UTR）的条件性诱变体系。在发育早期阻断微RNA-17~92与Bim的相互作用，可重现微RNA-17~92敲除所导致的致死性肺表型。由此可见，尽管存在数百个预测靶标，微RNA的关键功能可通过单个靶基因介导。我们从5份野生型胚胎第18.5天（E18.5）全肺组织及4份Bim 3'非翻译区突变纯合（Bim3'UTRmut/mut）的胚胎第18.5天全肺组织中提取总RNA。每份样本取100纳克总RNA作为起始材料，用于nCounter基因表达分析（nCounter gene expression analysis，NanoString Technologies公司）。