A better understanding of the liver’s response to toxic injury, which includes hepatocyte proliferation, and – unfortunately – an increased risk for hepatocellular carcinoma (HCC), is a prerequisite for the development of novel clinical treatments for chronic liver disease and improved cancer prevention. Existing drug therapies for HCC such as sorafenib extend patient survival by only three months. We recently developed a massively parallel in vivo screening platform to test the impact of genetic factors such as full-length cDNAs or miRNAs on liver repopulation and tumorigenesis. We have used this screening technology to build a map of all miRNAs active in liver regeneration. Here, we propose to exploit this innovative paradigm to conduct a comprehensive evaluation of the effects of the 135 most abundant but evolutionarily conserved hepatic miRNAs on the processes of recovery from toxic liver injury and HCC tumorigenesis. In Specific Aim 1, we will determine the combined benefits of three miRNAs identified in our prior screen on liver repopulation following toxic injuries, as a step toward using miRNA-mimetic drug therapies for liver diseases. This will be accomplished through delivery of miRNA-encoding plasmids or nanoparticles singly and in all combination. In Specific Aim 2, we will determine the impact of hepatic miRNAs and miRNA combinations on HCC tumor development in vivo. To this end, we have developed two models of rapid HCC development in mice, in which we will screen our library of 135 ‘tough decoys’ (“TuD’s”), or inhibitors of miRNA action, on tumor formation. We will quantify the abundance of all TuD’s using high throughput sequencing in the tumor-loaded liver compared to the input library. TuD’s enriched in after tumor formation target miRNAs that normally limit tumor growth, and those found less abundant target miRNAs that promote tumorigenesis. We will then test the combinations of the most potent miRNA effectors on tumor formation following systemic delivery. In Specific Aim 3 we will perform a conditional screen of miRNAs that impact Sorafenib resistance to identify novel combination treatments for the prevention or treatment of HCC. Together, our powerful genetic screens promise to identify miRNA effectors that can be employed for the treatment of acute liver injury and, in combination with Sorafenib, as a more effective treatment to prevent HCC initiation and progression.