"Project 2" Microprocessor overload in gamma-herpesviral oncogenesis

“项目 2” γ-疱疹病毒肿瘤发生中的微处理器过载

• 批准号: 10646232
• 负责人: ERIK K FLEMINGTON
• 金额: $ 28.48万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-09 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10646232
• 关键词: AIDS related cancer; AIDS-Related Lymphoma; Acquired Immunodeficiency Syndrome; Affinity; Animal Model; Autoimmune Diseases; Binding; Biogenesis; Bioinformatics; Biology; Burkitt Lymphoma; Cancer Patient; Cell Death; Cell Survival; Cells; Clinical; Collaborations; Cytoprotection; Data; Data Set; Down-Regulation; Epitopes; Epstein Barr Virus associated tumor; Epstein-Barr Virus Infections; Epstein-Barr Virus-Related Lymphoma; Epstein-Barr virus encoded RNA 1; Etiology; Gene Expression; Genes; Genetic Materials; HIV; Herpesviridae; Hodgkin Disease; Human Herpesvirus 4; Human Herpesvirus 8; Hybrids; Immune; Immune Targeting; Immune response; Infection; Innate Immune Response; Interferon Type II; Introns; Malignant Neoplasms; Mediating; Messenger RNA; MicroRNAs; Microprocessor; Modeling; Mus; Mutation; Nasopharynx Carcinoma; Non-Hodgkin' s Lymphoma; Oncogenes; Oncogenic; Pathway interactions; Patients; Phenotype; Production; Property; RNA; Regulatory Pathway; Resources; Risk; Role; SCID Mice; Sampling; Signal Pathway; Signal Transduction; Specimen; T-Cell Receptor; Testing; Time; Transcript; Transfection; Tumor Suppressor Proteins; Untranslated RNA; Viral; Viral Genes; Virus Diseases; Work; Xenograft procedure; adaptive immune response; biophysical analysis; cancer type; cell growth; co-infection; density; driver mutation; expression vector; gammaherpesvirus; humanized mouse; immune cell infiltrate; in vivo; malignant stomach neoplasm; mouse model; neoplastic cell; reconstitution; tissue culture; transcriptome; transcriptome sequencing; tumor; tumorigenesis; viral RNA; virus genetics

Summary The Epstein Barr virus (EBV) is a causal agent in Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, gastric cancer, nasopharyngeal carcinoma and autoimmune diseases. In the context of HIV co-infection (+ or - ART), EBV is especially problematic with a substantially elevated risk of EBV-associated lymphomas. The overall objective of this P01 is to investigate the role of non-coding RNAs in HIV-associated malignancies. Our assessment of EBV-associated Burkitt’s lymphoma (BL) and gastric cancer (GC) patient datasets revealed that outside of the ubiquitously expressed small EBER1/2 non-coding viral RNAs, in vivo viral gene expression is primarily limited to two overlapping long non-coding RNAs, A73 and RPMS1 and two large miRNA clusters contained within the RPMS1 introns. Strikingly, we found that viral miRNAs comprise up to 75% of all miRNAs expressed in the cell in both BL and GC clinical samples. MiRNA targetome analyses revealed that EBV (and KSHV (Project 1)) miRNAs interact more effectively with their targets than their cellular counterparts, in part through targeting more accessible regions of RNAs and through forming hybrids with more favorable binding energies. Functionally, we found that EBV miRNAs likely suppress innate and adaptive immune responses to viral infection. Together, these studies support the contention that EBV miRNAs are key contributors to the tumor phenotype in EBV associated lymphomas. Although certain cell miRNAs are known oncogenes, there is extensive evidence for a generalized lower expression of cell miRNAs in cancer. Further, driver mutations in miRNA processing genes have been identified in a number of cancer types. Utilizing patient RNA-seq data, we found that cell miRNA processing is diminished in EBV positive tumors and we show that expression of EBV miRNA clusters in EBV negative cells causes decreased processing of endogenous cell miRNAs. We hypothesize that the introduction of multiple copies of ectopic high-density miRNA clusters into cells upon EBV infection not only yields high viral miRNA production but also causes sequestration of microprocessor resources and inhibited processing of cell miRNAs. We further hypothesize that viral microprocessor inhibition diminishes the expression of cell tumor suppressor miRNAs to influence the tumor phenotype in a manner similar to genetic alterations of miRNA processing factor genes. In this proposal, we will use bench work, viral recombineering (Core C), and bioinformatics (Core B) in tissue culture, animal models (Core D), and clinical models to 1) determine the functional impact of direct viral miRNA targeting on immune regulatory pathways and 2) delineate the underlying mechanisms, the impacted oncogenic pathways, and the functional consequence of EBV-mediated microprocessor overload on the tumor phenotype. We will also begin preliminary studies to determine whether microprocessor overload is a shared feature extending to other oncogenic herpesviruses through testing the KSHV (Project 1) miRNA cluster.

概括 爱泼斯坦Barr病毒（EBV）是霍奇金淋巴瘤，非霍奇金淋巴瘤，胃肠道 癌症，鼻咽癌和自身免疫性疾病。在艾滋病毒共同感染的背景下（+或 - 艺术）， EBV尤其有问题，而与EBV相关淋巴瘤的风险显着升高。总体 该P01的目的是研究非编码RNA在与HIV相关的MALIGNANCYS中的作用。我们的 评估与EBV相关的伯基特淋巴瘤（BL）和胃癌（GC）患者数据集的评估表明， 在普遍表达的小EBER1/2非编码病毒RNA之外，体内病毒基因表达是 最初仅限于两个重叠的长无编码RNA，A73和RPMS1和两个大miRNA簇 包含在rpms1内含子中。令人惊讶的是，我们发现病毒miRNA最多占所有miRNA的75％ 在BL和GC临床样品中的细胞中表达。 miRNA目标分析表明EBV（和 KSHV（项目1））miRNA与靶标的相比，比其细胞对应物更有效地相互作用 通过靶向更容易访问的RNA区域，并通过形成具有更有利绑定的混合体 能量。在功能上，我们发现EBV miRNA可能会抑制先天和适应性免疫调查 病毒感染。这些研究共同支持以下论点 EBV相关淋巴瘤中的肿瘤表型。 尽管某些细胞miRNA是已知的肿瘤基因，但仍有广泛的证据表明较低 细胞miRNA在癌症中的表达。此外，miRNA加工基因中的驱动突变已经 在多种癌症类型中鉴定出来。利用患者RNA-seq数据，我们发现细胞miRNA处理是 在EBV阳性肿瘤中减少，我们表明EBV miRNA簇在EBV阴性细胞中的表达 导致内源细胞miRNA的加工减少。我们假设引入多个 EBV感染后的Ecopic高密度miRNA簇进入细胞的副本不仅产生高病毒miRNA 生产但还会引起微处理器资源的会议并抑制细胞的处理 mirnas。我们进一步假设病毒微处理器抑制会减少细胞肿瘤的表达 抑制miRNA以类似于miRNA的遗传改变的方式影响肿瘤表型 处理因子基因。 在此提案中，我们将使用台式工作，病毒重组（核心C）和组织中的生物信息学（核心B） 培养，动物模型（核心D）和临床模型至1）确定直接病毒miRNA的功能影响 靶向免疫调节途径，2）描述了基本机制，受影响 致癌途径以及EBV介导的微处理器超负荷肿瘤上的功能后果 表型。我们还将开始初步研究，以确定微处理器超负荷是否共享 通过测试KSHV（项目1）miRNA簇扩展到其他致癌疱疹病毒的特征。