Restoration of RNA helicase DDX5 suppresses hepatitis B virus (HBV) biosynthesis and Wnt signaling in HBV-related hepatocellular carcinoma

Abstract

<b>Rationale:</b> RNA helicase DDX5 is downregulated during hepatitis B virus (HBV) replication, and poor prognosis HBV-related hepatocellular carcinoma (HCC). The aim of this study is to determine the mechanism and significance of DDX5 downregulation for HBV-driven HCC, and identify biologics to prevent DDX5 downregulation. <b>Methods:</b> Molecular approaches including immunoblotting, qRT-PCR, luciferase transfections, hepatosphere assays, Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq), and RNA-seq were used with cellular models of HBV replication, HBV infection, and HBV-related liver tumors, as well as bioinformatic analyses of liver cancer cells from two independent cohorts. <b>Results:</b> We demonstrate that HBV infection induces expression of the proto-oncogenic miR17~92 and miR106b~25 clusters which target the downregulation of DDX5. Increased expression of these miRNAs is also detected in HBV-driven HCCs exhibiting reduced <i>DDX5</i> mRNA. Stable DDX5 knockdown (DDX5^KD) in HBV replicating hepatocytes increased viral replication, and resulted in hepatosphere formation, drug resistance, Wnt activation, and pluripotency gene expression. ATAC-seq of DDX5^KD compared to DDX5 wild-type (WT) cells identified accessible chromatin regions enriched in regulation of Wnt signaling genes. RNA-seq analysis comparing WT versus DDX5^KD cells identified enhanced expression of multiple genes involved in Wnt pathway. Additionally, expression of <i>Disheveled</i>, <i>DVL1</i>, a key regulator of Wnt pathway activation, was significantly higher in liver cancer cells with low <i>DDX5</i> expression, from two independent cohorts. Importantly, inhibitors (antagomirs) to miR17~92 and miR106b~25 restored DDX5 levels, reduced <i>DVL1</i> expression, and suppressed both Wnt activation and viral replication. <b>Conclusion</b><i>:</i> DDX5 is a negative regulator of Wnt signaling and hepatocyte reprogramming in HCCs. Restoration of DDX5 levels by miR17~92 / miR106b~25 antagomirs in HBV-infected patients can be explored as both antitumor and antiviral strategy.