"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: 10403016
• 负责人: ERIK K FLEMINGTON
• 金额: $ 28.04万
• 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/10403016
• 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; Cells; Clinical; Collaborations; Data; Data Set; Down-Regulation; Epitopes; Epstein Barr Virus associated tumor; Epstein-Barr Virus Infections; 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; Infiltration; Interferon Type II; Introns; Lymphoma; 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; base; biophysical analysis; cancer type; cell growth; co-infection; density; driver mutation; expression vector; gammaherpesvirus; humanized mouse; in vivo; malignant stomach neoplasm; mouse model; neoplastic cell; pri-miRNA; 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 EB 病毒 (EBV) 是霍奇金淋巴瘤、非霍奇金淋巴瘤、胃淋巴瘤的致病因子。 在 HIV 合并感染（+ 或 - ART）的情况下，癌症、鼻咽癌和自身免疫性疾病。 EB 病毒尤其成问题，因为 EB 病毒相关淋巴瘤的风险显着升高。 本 P01 的目标是研究非编码 RNA 在 HIV 相关恶性肿瘤中的作用。 对 EB 病毒相关伯基特淋巴瘤 (BL) 和胃癌 (GC) 患者数据集的评估表明， 除了普遍表达的小 EBER1/2 非编码病毒 RNA 之外，体内病毒基因表达是 主要限于两个重叠的长非编码RNA，A73和RPMS1以及两个大的miRNA簇 令人惊讶的是，我们发现病毒 miRNA 占所有 miRNA 的 75%。 在 BL 和 GC 临床样本的细胞中表达的 MiRNA 靶标组分析表明 EBV（和）。 KSHV（项目 1））部分 miRNA 与靶标的相互作用比与细胞器官的相互作用更有效 通过靶向更容易接近的 RNA 区域并通过具有更有利结合的杂交体形成 从功能上讲，我们发现 EBV miRNA 可能会抑制先天性和适应性免疫反应。 这些研究共同支持了 EBV miRNA 是病毒感染的关键因素这一论点。 EBV 相关淋巴瘤的肿瘤表型。 尽管某些细胞 miRNA 是已知的癌基因，但有大量证据表明普遍较低的致癌基因。 此外，miRNA 加工基因的驱动突变已被证实。 利用患者 RNA-seq 数据，我们发现细胞 miRNA 处理是在多种癌症类型中发现的。 EBV 阳性肿瘤中 EBV miRNA 簇的表达减少，并且我们发现 EBV 阴性细胞中 EBV miRNA 簇的表达 导致内源细胞 miRNA 的加工减少。 EBV 感染后异位高密度 miRNA 簇的拷贝进入细胞不仅产生高病毒 miRNA 生产，但也会导致微处理器资源的隔离并抑制单元的处理 我们进一步发现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 遗传改变的方式影响肿瘤表型 加工因子基因。 在本提案中，我们将在组织中使用实验室工作、病毒重组工程（核心 C）和生物信息学（核心 B） 培养、动物模型（核心 D）和临床模型，以 1) 确定直接病毒 miRNA 的功能影响 针对免疫调节途径，2）描述潜在机制、影响 致癌途径，以及 EBV 介导的微处理器过载对肿瘤的功能性影响 我们还将开始初步研究以确定微处理器过载是否是一个共同的表型。 通过测试 KSHV（项目 1）miRNA 簇，该功能扩展到其他致癌疱疹病毒。