LAUSR

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 following toxic liver injury using Fah-/- mice. METHODS We constructed plasmid pools encoding more than 30,000 tough decoy iRNA inhibitors (hair-pin nucleic acids designed to specifically inhibit interactions between miRNAs s and their targets) to target hepatocyte miRNAs in a pairwise manner. The plasmid libraries were delivered to hepatocytes in Fah-/- mice at time of liver injury via hydrodynamic tail vein injection. Integrated transgene-containing transposons were quantified following liver repopulation via high-throughput sequencing. Changes in polysome-bound transcripts following 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 lost or gained following 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 required for liver regeneration after injury in live mice.