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.

摘要 该建议研究了RNA解旋酶DDX5/p68在调节免疫信号通路和 基因表达与乙肝病毒感染和乙肝病毒相关性肝细胞癌 (肝细胞癌)。我们早期的研究表明，DDX5是一种宿主限制因子，通过一种 机制尚不清楚，DDX5在乙肝相关性肝癌中表达下调，预后不良，在 晚期肝细胞癌对多激酶抑制剂(MTKIs)的反应，以及DDX5对照 STAT1翻译。我们对表现出DDX5基因敲除的人肝癌细胞系的RNAseq分析发现 激活Wnt和非典型的NF-κB信号，作为DDX5的下游靶标。 从病毒生物合成和肝癌的角度进一步了解DDX5的作用机制 在致病机制方面，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伽马诱导蛋白16(IFI16)，一种核DNA传感器和 核内复制病毒的限制因子，作为最显著的细胞因子与 DDX5。最近的一项研究表明，IFI16与HBV微染色体结合导致对HBV病毒的抑制 复制。然而，作为逃避天然免疫监视的一种策略，乙肝病毒下调IFI16的表达 在慢性乙肝病毒感染患者中。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 基于天然核提取物的层析和随后的无标记定量MS图谱的结果显示 DDX5和IFI16与辅助和核心PRC2亚基HDAC1、2和DNMT1共洗脱。这是一致的 根据Corum数据库的预测，DDX5/IFI16与MeCP1复合体相互作用，包括 组蛋白脱乙酰基酶(HDACs)和DNA甲基转移酶(DNMTs)及染色质沉默多聚体 抑制性复合体2(PRC2)。重要的是，用重组核酸酶苯甲酸酶处理核裂解产物 DDX5与IFI16的结合减少，表明RNA参与了该复合体的形成。 然而，参与DDX5/IFI16复合体形成的RNA底物目前尚不清楚。 基于这些初步观察，我们假设DDX5/IFI16复合体抑制病毒 转录，以及肝细胞癌进展过程中重要基因的细胞转录。要解决这一假设 我们针对这一机制的关键方面提出了两个独立的具体目标： 目的：研究表观遗传性DDX5/IFI16沉默复合体的抗病毒作用。 目的：探讨DDX5/IFI16复合体在肝癌基因表达中的作用。 影响：该项目的成功完成将确定作为治疗目标的基本机制 沉默HBV微染色体转录，逆转细胞基因表达变化 与预后不良有关的乙肝病毒相关性肝癌。