A High-Content Screen Identifies MicroRNAs That Regulate Liver Repopulation After Injury in Mice

• Published in: Gastroenterology (New York, N.Y. 1943) Vol. 158; no. 4; pp. 1044 - 1057.e17
• Main Authors: Zahm, Adam M., Wang, Amber W., Wang, Yue J., Schug, Jonathan, Wangensteen, Kirk J., Kaestner, Klaus H.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2020
• Subjects: Animals; Chromosome Mapping; Genetic Screen; Hepatocytes - physiology; High-Throughput Nucleotide Sequencing - methods; Hydrolases - deficiency; Liver - injuries; Liver - physiopathology; Liver Regeneration - genetics; Mice; microRNA; MicroRNAs - analysis; MicroRNAs - antagonists & inhibitors; Plasmids; Post-Transcriptional Regulation; RNA-Binding Proteins - analysis; Tough decoy; RISC; TuD; pT; TuDstd; mRNA; TRAP; TuD374; FAH; Genetic Screen; Tough decoy; Post-Transcriptional Regulation; TuDctrl; UTR; TuDtail; AGO2; microRNA; miRNA; MI; Seq; gDNA; MBS
• ISSN: 0016-5085; 1528-0012
• DOI: 10.1053/j.gastro.2019.11.025

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. 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. 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. We developed a screen to identify miRNAs that regulate liver repopulation after injury in live mice.