Role of DDX5 in Hepatitis B virus transcription and hepatocarcinogenesis

DDX5在乙型肝炎病毒转录和肝癌发生中的作用

• 批准号: 10665448
• 负责人: Ourania M. Andrisani
• 金额: $ 22.73万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-16 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10665448
• 关键词: Address; Anabolism; Antiviral Agents; Binding; Cancer cell line; Cell Nucleus; Chromatin; Chromatography; Chronic; Chronic Hepatitis B; Circular DNA; Complex; DNA; Data; Databases; Development; Down-Regulation; EZH2 gene; Epigenetic Process; Etiology; Exhibits; Gene Expression; Gene Silencing; Genes; Genetic Transcription; HDAC1 gene; Hepatitis B Infection; Hepatitis B Vaccines; Hepatitis B Virus; Hepatocarcinogenesis; Hepatocyte; Histone Deacetylase; Human; Immune signaling; Immunologic Surveillance; In Vitro; Individual; Interferon Type II; Interferon alpha; Interferons; Label; Link; Malignant neoplasm of liver; Mediating; Methyltransferase; Modeling; Nuclear; Nuclear Extract; Oncogenes; Patients; Polycomb; Primary carcinoma of the liver cells; Prognosis; Proteins; Proteomics; RNA; RNA Helicase; Recombinants; Repression; Role; STAT1 gene; Signal Pathway; Signal Transduction; Transferase; Translations; Viral; Virus; Virus Diseases; Virus Replication; WNT Signaling Pathway; chronic infection; curative treatments; effective therapy; epigenetic silencing; helicase; insight; kinase inhibitor; knock-down; novel; nuclease; response; sensor; therapeutic target; transcription factor; transcriptome sequencing

Abstract This proposal investigates the role of the RNA helicase DDX5/p68 in regulating immune signaling pathways and gene expression, as it relates to hepatitis B virus (HBV) infection and HBV-related hepatocellular carcinoma (HCC). Our earlier studies demonstrated that DDX5 is a host restriction factor in HBV biosynthesis by a mechanism not yet understood, that DDX5 is downregulated in HBV-related HCC with poor prognosis and in response to multi-kinase inhibitors (mTKIs) used for the treatment of advanced HCC, and that DDX5 controls STAT1 translation. Our RNAseq analyses of human liver cancer cell lines exhibiting DDX5 knockdown, identified activation of Wnt and non-canonical NF-κB signaling, as downstream targets of DDX5. To gain further insight into the mechanism of DDX5 action in terms of virus biosynthesis and liver cancer pathogenesis, we performed a proteomics (LC-MS/MS) study to identify cellular interacting partners of DDX5. Surprisingly, our studies identified the Interferon gamma induced protein 16 (IFI16), a nuclear DNA sensor and restriction factor for viruses that replicate in the nucleus, as the most prominent cellular factor interacting with DDX5. A recent study has demonstrated that IFI16 binds the HBV minichromosome leading to inhibition of HBV replication. However, HBV downregulates IFI16 expression as a strategy to escape innate immune surveillance in chronically HBV infected patients. Significantly, the role of DDX5 in this interaction is currently unknown. Our results based on chromatography of native nuclear extracts followed by label-free quantitative MS profiling show that DDX5 and IFI16 co-eluted with auxiliary and core PRC2 subunits, HDAC1, 2 and DNMT1. This is consistent with the prediction using the CORUM database, that DDX5/IFI16 interact with the MeCP1 complex, comprised of histone deacetylases (HDACs) & DNA methyl transferases (DNMTs), and the chromatin silencing Polycomb Repressive Complex 2 (PRC2). Importantly, treatment of nuclear lysates with recombinant nuclease benzonase reduced association of DDX5 with IFI16, suggesting involvement of an RNA in the formation of this complex. However, the RNA substrate involved in formation of the DDX5/IFI16 complex is currently unknown. Based on these preliminary observations, we hypothesize that the DDX5/IFI16 complex represses viral transcription, as well as cellular transcription of genes important for HCC progression. To address this hypothesis we propose two independent specific aims focusing on key aspects of this mechanism: Aim1: To determine the antiviral effect of the epigenetic DDX5/IFI16 silencing complex. Aim2: To determine the role of the DDX5/IFI16 complex in the expression of liver cancer genes. Impact: Successful completion of this project will identify an essential mechanism as a therapeutic target for silencing transcription from the HBV minichromosome, and reversing cellular gene expression changes associated with poor prognosis HBV-related liver cancer.

抽象的 该提案研究了 RNA 解旋酶 DDX5/p68 在调节免疫信号通路和 基因表达，因为它与乙型肝炎病毒 (HBV) 感染和 HBV 相关肝细胞癌有关 （肝癌）。我们早期的研究表明，DDX5 是 HBV 生物合成的宿主限制因子。 机制尚不清楚，DDX5 在 HBV 相关 HCC 中下调，预后不良，并且在 对用于治疗晚期 HCC 的多激酶抑制剂 (mTKI) 的反应，DDX5 控制 STAT1 翻译。我们对表现出 DDX5 敲低的人类肝癌细胞系进行 RNAseq 分析，发现 激活 Wnt 和非经典 NF-κB 信号传导，作为 DDX5 的下游靶标。 进一步深入了解DDX5在病毒生物合成和肝癌方面的作用机制 发病机制中，我们进行了蛋白质组学 (LC-MS/MS) 研究，以确定 DDX5 的细胞相互作用伙伴。 令人惊讶的是，我们的研究发现了干扰素 γ 诱导蛋白 16 (IFI16)，一种核 DNA 传感器和 病毒在细胞核中复制的限制因子，作为与病毒相互作用的最重要的细胞因子 DDX5。最近的一项研究表明，IFI16 结合 HBV 微小染色体，从而抑制 HBV 复制。然而，HBV 下调 IFI16 表达作为逃避先天免疫监视的策略 慢性乙型肝炎病毒感染患者。值得注意的是，DDX5 在这种相互作用中的作用目前尚不清楚。我们的 基于天然核提取物色谱和无标记定量 MS 分析的结果显示 DDX5 和 IFI16 与辅助和核心 PRC2 亚基、HDAC1、2 和 DNMT1 共同洗脱。这是一致的 使用 CORUM 数据库预测，DDX5/IFI16 与 MeCP1 复合物相互作用，包括 组蛋白脱乙酰酶 (HDAC) 和 DNA 甲基转移酶 (DNMT) 以及染色质沉默 Polycomb 压抑情结 2 (PRC2)。重要的是，用重组核酸酶 Benzonase 处理核裂解物 DDX5 与 IFI16 的关联性降低，表明 RNA 参与了该复合物的形成。 然而，目前尚不清楚参与 DDX5/IFI16 复合物形成的 RNA 底物。 基于这些初步观察，我们假设 DDX5/IFI16 复合物抑制病毒 转录，以及对 HCC 进展重要的基因的细胞转录。为了解决这个假设 我们提出了两个独立的具体目标，重点关注该机制的关键方面： 目的1：确定表观遗传DDX5/IFI16沉默复合物的抗病毒作用。 目的2：确定DDX5/IFI16复合物在肝癌基因表达中的作用。 影响：该项目的成功完成将确定一个重要的机制作为治疗靶点 沉默 HBV 微小染色体的转录，并逆转细胞基因表达变化 与 HBV 相关肝癌预后不良有关。