A high-content in vivo screen to identify microRNA epistasis in the repopulating mouse liver

Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing; yet the roles of individual miRNAs or their combinatorial effects in this process are largely unknown. In this study, we sought to identify miRNAs that regulate hepatocyte repopulation following toxic liver injury in a high-throughput manner using the Fah-/- mouse. We constructed plasmid pools encoding over 30,000 tough decoy (TuD) miRNA inhibitors designed to target hepatocyte miRNAs in a pairwise manner. Plasmid libraries were delivered to hepatocytes of Fah-/- mice at the time of liver injury via hydrodynamic tail vein injection and integrated transgene-containing transposons were quantified following 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. Analysis of TuD abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly altered following repopulation. We classified a subset of miRNA-binding sites (MBSs) as having strong effect on liver repopulation, thus implicating the targeted hepatocyte miRNAs as regulators of this process. Furthermore, we generated a high-content map of pairwise interactions between 171 MBSs and identified both synergistic and redundant effects. Our study highlights the power of higher-order screens to uncover miRNA functions that would go undetected by individual miRNA perturbations, and provides a new paradigm for the study of epistasis of miRNA activities.