Theranostics 2020; 10(24):10957-10972. doi:10.7150/thno.49629

Research Paper

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

Saravana Kumar Kailasam Mani1,4*, Bingyu Yan2,4*, Zhibin Cui1,4, Jiazeng Sun1,4, Sagar Utturkar4, Adrien Foca5, Nadim Fares6, David Durantel5, Nadia Lanman4, Philippe Merle6, Majid Kazemian2,3,4✉, Ourania Andrisani1,4✉

1. Department of Basic Medical Sciences.
2. Department of Biochemistry.
3. Department of Computer Science.
4. Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907.
5. Cancer Research Center of Lyon UMR Inserm 1052 - CNRS 5286.
6. Department of Hepatology, Hôpital de la Croix-Rousse, Hospices Civils de Lyon, Université Lyon 1, Lyon, France
*equal contribution

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Citation:
Mani SKK, Yan B, Cui Z, Sun J, Utturkar S, Foca A, Fares N, Durantel D, Lanman N, Merle P, Kazemian M, Andrisani O. Restoration of RNA helicase DDX5 suppresses hepatitis B virus (HBV) biosynthesis and Wnt signaling in HBV-related hepatocellular carcinoma. Theranostics 2020; 10(24):10957-10972. doi:10.7150/thno.49629. Available from http://www.thno.org/v10p10957.htm

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Abstract

Rationale: 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.

Methods: 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.

Results: 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 DDX5 mRNA. Stable DDX5 knockdown (DDX5KD) in HBV replicating hepatocytes increased viral replication, and resulted in hepatosphere formation, drug resistance, Wnt activation, and pluripotency gene expression. ATAC-seq of DDX5KD compared to DDX5 wild-type (WT) cells identified accessible chromatin regions enriched in regulation of Wnt signaling genes. RNA-seq analysis comparing WT versus DDX5KD cells identified enhanced expression of multiple genes involved in Wnt pathway. Additionally, expression of Disheveled, DVL1, a key regulator of Wnt pathway activation, was significantly higher in liver cancer cells with low DDX5 expression, from two independent cohorts. Importantly, inhibitors (antagomirs) to miR17~92 and miR106b~25 restored DDX5 levels, reduced DVL1 expression, and suppressed both Wnt activation and viral replication.

Conclusion: 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.

Keywords: Hepatitis B virus, Hepatocellular Carcinoma, RNA helicase DDX5, miR17~92/miR106b~25 & antagomirs, Wnt/β-catenin signaling.