Regulation of viral replication by microRNAs (miRNAs) and the role of microRNAs in cancer are two frontiers of research that have been under-investigated in the past. This research area has significant promise to greatly expand our molecular understanding of host-viral interactions and the process of malignant transformation. In the past year, our laboratory has pioneered studies on miRNAs in hepatocarcinogenesis and HBV replication. These investigations have identified miRNAs that are highly overexpressed or underexpressed in HCC, and identified candidate miRNAs that affect HBV replication. Several hypotheses regarding mechanisms of miRNA involvement in hepatocarcinogenesis and viral replication have arisen. The specific aims are: Aim 1. Investigate the effects of cellular miRNAs on HBV replication. We will test the hypothesis that cellular miRNAs act as cellular "rheostats" that regulate the level of HBV replication. Our first studies will focus on miR-16, since it has a target site that is conserved among HBV genomes and supporting data suggest it is a negative regulator for HBV replication. Aim 2. We will investigate the effects of miRNA on the early phases of HBV replication. We will test the hypothesis that cellular miRNAs with HBV target sites can inhibit or block early phases of HBV replication. Aim 3: We will investigate the role of miRNAs in hepatocarcinogenesis by testing how knocking out or inducing miRNAs affect the malignant HCC phenotype. Aim 4: We will identify miRNA targets in cancer cells and primary tumors, using a novel cloning/sequencing approach based on immunoprecipitation of RISC that contain miRNAs and their targets.