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A High-Content Screen Identifies MicroRNAs That Regulate Liver Repopulation After Injury in Mice.
Gastroenterology ( IF 29.4 ) Pub Date : 2019-11-20 , DOI: 10.1053/j.gastro.2019.11.025
Adam M Zahm 1 , Amber W Wang 1 , Yue J Wang 2 , Jonathan Schug 1 , Kirk J Wangensteen 1 , Klaus H Kaestner 1
Affiliation  

BACKGROUND & AIMS Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing, but it is not clear which miRNAs regulate this process. We developed a high-throughput screen to identify miRNAs that regulate hepatocyte repopulation after toxic liver injury using fumarylacetoacetate hydrolase-deficient mice. METHODS We constructed plasmid pools encoding more than 30,000 tough decoy miRNA inhibitors (hairpin nucleic acids designed to specifically inhibit interactions between miRNAs and their targets) to target hepatocyte miRNAs in a pairwise manner. The plasmid libraries were delivered to hepatocytes in fumarylacetoacetate hydrolase-deficient mice at the time of liver injury via hydrodynamic tail-vein injection. Integrated transgene-containing transposons were quantified after liver repopulation via high-throughput sequencing. Changes in polysome-bound transcripts after miRNA inhibition were determined using translating ribosome affinity purification followed by high-throughput sequencing. RESULTS Analyses of tough decoy abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly depleted or increased after repopulation. We classified a subset of miRNA binding sites as those that have strong effects on liver repopulation, implicating the targeted hepatocyte miRNAs as regulators of this process. We then generated a high-content map of pairwise interactions between 171 miRNA-binding sites and identified synergistic and redundant effects. CONCLUSIONS We developed a screen to identify miRNAs that regulate liver repopulation after injury in live mice.

中文翻译:

一个高内涵的屏幕可以识别在小鼠受伤后调节肝脏再增殖的MicroRNA。

背景与目的在microRNA(miRNA)加工中具有肝细胞特异性缺陷的小鼠中,肝脏的再生受到损害,但尚不清楚哪些miRNA调控这一过程。我们开发了一种高通量的筛选方法,以鉴定使用富马酰乙酰乙酸水解酶缺陷型小鼠在毒性肝损伤后可调节肝细胞重新聚集的miRNA。方法我们构建了质粒库,该质粒库以成对方式靶向肝细胞miRNA,编码超过30,000种坚韧的诱饵miRNA抑制剂(设计为特异性抑制miRNA与它们的靶标之间相互作用的发夹核酸)。在肝损伤时,通过流体动力尾静脉注射将质粒文库递送至富马酸乙酰乙酸酯水解酶缺陷型小鼠中的肝细胞。通过高通量测序,在肝脏重新组装后,对整合的含转基因的转座子进行了定量。使用翻译核糖体亲和纯化,然后进行高通量测序,确定miRNA抑制后多核糖体结合的转录物的变化。结果肝细胞基因组DNA和输入质粒库中艰难诱饵的丰度分析确定了数千种miRNA抑制剂,这些抑制剂在重新种群后显着减少或增加。我们将miRNA结合位点的一个子集归类为对肝脏重生有强烈影响的位点,暗示目标肝细胞miRNAs是该过程的调节剂。然后,我们生成了171个miRNA结合位点之间的成对相互作用的高内涵图,并确定了协同效应和冗余效应。
更新日期:2019-11-21
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