Department of Basic Medical Sciences.
Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907.
Theranostics. 2020 Sep 1;10(24):10957-10972. doi: 10.7150/thno.49629. eCollection 2020.
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. 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. We demonstrate that HBV infection induces expression of the proto-oncogenic miR1792 and miR106b25 clusters which target the downregulation of DDX5. Increased expression of these miRNAs is also detected in HBV-driven HCCs exhibiting reduced mRNA. Stable DDX5 knockdown (DDX5) in HBV replicating hepatocytes increased viral replication, and resulted in hepatosphere formation, drug resistance, Wnt activation, and pluripotency gene expression. ATAC-seq of DDX5 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 cells identified enhanced expression of multiple genes involved in Wnt pathway. Additionally, expression of , , a key regulator of Wnt pathway activation, was significantly higher in liver cancer cells with low expression, from two independent cohorts. Importantly, inhibitors (antagomirs) to miR1792 and miR106b25 restored DDX5 levels, reduced expression, and suppressed both Wnt activation and viral replication. DDX5 is a negative regulator of Wnt signaling and hepatocyte reprogramming in HCCs. Restoration of DDX5 levels by miR1792 / miR106b25 antagomirs in HBV-infected patients can be explored as both antitumor and antiviral strategy.
RNA 解旋酶 DDX5 在乙型肝炎病毒 (HBV) 复制和预后不良的 HBV 相关肝细胞癌 (HCC) 中下调。本研究旨在确定 DDX5 下调对 HBV 驱动的 HCC 的机制和意义，并确定预防 DDX5 下调的生物制剂。 我们使用 HBV 复制、HBV 感染和 HBV 相关肝癌的细胞模型以及两个独立队列的肝癌细胞的生物信息学分析，采用包括免疫印迹、qRT-PCR、荧光素酶转染、肝细胞球体测定、转座酶可及染色质测序 (ATAC-seq) 和 RNA-seq 在内的分子方法。 我们证明 HBV 感染诱导原癌基因 miR1792 和 miR106b25 簇的表达，这些 miRNA 靶向 DDX5 的下调。在表现出 mRNA 减少的 HBV 驱动 HCC 中也检测到这些 miRNA 的表达增加。在 HBV 复制的肝细胞中稳定敲低 DDX5 (DDX5) 会增加病毒复制，并导致肝细胞球体形成、耐药性、Wnt 激活和多能性基因表达。与 DDX5 野生型 (WT) 细胞相比，DDX5 的 ATAC-seq 鉴定了富含 Wnt 信号基因调节的可及染色质区域。比较 WT 与 DDX5 细胞的 RNA-seq 分析鉴定出多个参与 Wnt 途径的基因表达增强。此外，两个独立队列的肝癌细胞中，关键的 Wnt 途径激活调节剂 的表达明显更高。重要的是，miR1792 和 miR106b25 的抑制剂 (antagomirs) 可恢复 DDX5 水平，降低 表达，并抑制 Wnt 激活和病毒复制。 DDX5 是 HCC 中 Wnt 信号和肝母细胞重编程的负调节剂。在 HBV 感染患者中，通过 miR1792 / miR106b25 antagomirs 恢复 DDX5 水平可以作为抗肿瘤和抗病毒策略进行探索。
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