MicroRNA regulation of human T and B cell activation

MicroRNA 调控人类 T 细胞和 B 细胞活化

• 批准号: 8212134
• 负责人: Daniel R. Salomon
• 金额: $ 46.53万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8212134
• 关键词: 3' Untranslated Regions; Animals; B-Cell Activation; B-Lymphocytes; Back; Binding Proteins; Biological Markers; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Cell Lineage; Cell Nucleus; Cell physiology; Cells; Clinical; Cytoplasm; Data; Databases; Development; Dicer Enzyme; Drug Delivery Systems; Enzymes; Evolution; Exons; Functional RNA; Gene Expression; Gene Expression Profiling; Gene Silencing; Gene Targeting; Generations; Genes; Genetic Transcription; Genome; Genomics; Heart; Hematopoietic; Human; Immune; Immune response; Immunity; Immunology; Immunosuppression; Individual; Link; Literature; Lymphocyte; Lymphocyte Activation; Maps; Mediating; Messenger RNA; Methods; MicroRNAs; Modeling; Molecular; Mus; Nucleotides; Organism; Parents; Pathway interactions; Play; Post-Transcriptional Regulation; Principal Investigator; Process; Protein Biosynthesis; Protein Synthesis Inhibition; Proteins; Proteomics; Regulation; Review Literature; Role; Sampling; Seeds; Sequence Homologs; Source; Structure; Systems Biology; T-Lymphocyte; Testing; Time; Tissues; Transcript; Transcriptional Regulation; Translating; Translational Repression; Translations; Transplant Recipients; Transplantation; Transplantation Immunology; Work; base; cardiogenesis; cell type; embryonic stem cell; immune activation; mRNA Expression; mRNA Transcript Degradation; next generation; novel; programs; promoter; public health relevance; research study; stem; tandem mass spectrometry; transcription factor

DESCRIPTION (provided by applicant): Non-coding RNAs (ncRNAs) are not translated into protein but are functional in regulation of transcription. There is strong evidence that ncRNAs are key players in developmental, cellular, and immunological processes. One class of ncRNAs are microRNAs (miRNAs), discovered in 1993, that are believed to regulate almost 30% of all human genes. Each miRNA has a few to as many as 200 target genes, which they modulate by inhibiting mRNA at a transcriptional level (mRNA degradation) or the translational level (resulting in inhibition of protein synthesis). There are now over 670 known human miRNAs (Sanger miRBase) though only a few are mechanistically validated. Current literature identifies 30 miRNAs associated with hematopoietic lineage cells but only a small subset documented in T and B cells. We hypothesize that miRNAs play a pivotal role in regulation of human immunity by targeting key differentially and constitutively expressed genes regulating T and B lymphocyte activation, differentiation and survival. Despite many studies describing immune-associated transcripts and proteins orchestrating the transplant immune response, little is known about regulating this genomic complexity. The premise of this proposal is that identifying the miRNA- regulated molecular networks within this complexity will illuminate the key pathways in a way that cannot be achieved by simple gene expression profiling or proteomics. To test this premise in our preliminary studies we used Whole Exon Arrays for mRNA and a novel stem-loop qPCR method for 420 known miRNAs to study human T and B cell activation. Results revealed 62 differentially expressed miRNAs of which 42 have not previously been linked to lymphocyte or hematopoietic lineage regulation, over 3,000 differentially expressed mRNAs, unique miRNA profiles distinguishing T and B cell activation and a number of high value candidates for miRNA regulation of biologically significant molecular pathways. The objective of this proposal is to complete a full analysis of miRNA expression in human T and B cell activation, to investigate the novel possibility that post-transcriptional processing of precursors for miRNA in the nucleus is important, to use tandem mass spectrometry proteomics and Next Generation deep sequencing (Roche 454 FLX) to discover the correlations between protein, mRNA and miRNA expression and molecular network regulation during activation, and to validate a number of high value miRNA candidates in biologically significant pathways. If our multidimensional "omics" approach is successful, then it is a proof of concept for an effort to organize the full force of a large collaborative group at validating all the major pathways for each cell type, identifying the impact of immunosuppression, correlate results with clinical samples from transplant patients and aim to advance our understanding of transplant immunology from the level of individual gene-linked mechanisms to integrated molecular networks. PUBLIC HEALTH RELEVANCE: MicroRNAs are a potentially exciting class of cell process regulators that work by inhibiting the synthesis of proteins. Our preliminary results demonstrate that over 60 microRNAs are involved in the activation of human lymphocytes during immune activation. The objective of the present work is to apply a multi-dimensional systems biology or "omic" approach including gene profiling, proteomics and Next Generation sequencing to understanding the mechanisms and pathways regulated by microRNAs in the human immune response. The challenge is to develop and validate new strategies that can reduce the current complexity of gene expression and proteomic data, back down to discrete molecular networks and validate these in hypothesis-driven, mechanism-based experiments. When this evolution is accomplished, we will have a new understanding of transplantation immunology, discover a new generation of biomarkers and identify the next generation of potential drug targets.

描述（由申请人提供）：非编码 RNA (ncRNA) 不翻译成蛋白质，但在转录调节中发挥作用。有强有力的证据表明 ncRNA 在发育、细胞和免疫过程中发挥着关键作用。一类 ncRNA 是 1993 年发现的 microRNA (miRNA)，据信它调节着近 30% 的人类基因。每个 miRNA 都有几个到多达 200 个目标基因，它们通过在转录水平（mRNA 降解）或翻译水平（导致蛋白质合成抑制）抑制 mRNA 来调节这些基因。目前已知的人类 miRNA (Sanger miRBase) 超过 670 种，但只有少数经过机制验证。目前的文献鉴定了 30 种与造血谱系细胞相关的 miRNA，但只有一小部分记录在 T 和 B 细胞中。我们假设 miRNA 通过靶向调节 T 和 B 淋巴细胞活化、分化和存活的关键差异和组成型表达基因，在人类免疫调节中发挥关键作用。尽管许多研究描述了协调移植免疫反应的免疫相关转录本和蛋白质，但人们对调节这种基因组复杂性知之甚少。该提案的前提是，在这种复杂性中识别 miRNA 调节的分子网络将以简单的基因表达谱或蛋白质组学无法实现的方式阐明关键途径。为了在我们的初步研究中测试这一前提，我们使用了 mRNA 的全外显子阵列和针对 420 种已知 miRNA 的新型茎环 qPCR 方法来研究人类 T 和 B 细胞的激活。结果揭示了 62 个差异表达的 miRNA，其中 42 个以前未与淋巴细胞或造血谱系调节相关，超过 3,000 个差异表达的 mRNA，区分 T 和 B 细胞激活的独特 miRNA 谱，以及许多用于 miRNA 调节具有生物学意义的分子的高价值候选者。途径。本提案的目的是完成对人类 T 和 B 细胞激活中 miRNA 表达的全面分析，研究细胞核中 miRNA 前体转录后加工的重要性，利用串联质谱蛋白质组学和 Next新一代深度测序 (Roche 454 FLX) 发现蛋白质、mRNA 和 miRNA 表达与激活过程中分子网络调控之间的相关性，并在具有生物学意义的途径中验证许多高价值 miRNA 候选者。如果我们的多维“组学”方法成功，那么它就是一个概念证明，可以组织一个大型协作小组的全部力量来验证每种细胞类型的所有主要途径，识别免疫抑制的影响，将结果与来自移植患者的临床样本，旨在增进我们对移植免疫学的理解，从个体基因相关机制到整合分子网络的水平。 公共健康相关性：MicroRNA 是一类潜在的令人兴奋的细胞过程调节剂，通过抑制蛋白质的合成发挥作用。我们的初步结果表明，超过 60 个 microRNA 参与免疫激活过程中人类淋巴细胞的激活。目前工作的目标是应用多维系统生物学或“组学”方法，包括基因分析、蛋白质组学和下一代测序，以了解人类免疫反应中 microRNA 调节的机制和途径。我们面临的挑战是开发和验证新的策略，以降低当前基因表达和蛋白质组数据的复杂性，回到离散的分子网络，并在假设驱动、基于机制的实验中验证这些网络。当这一进化完成时，我们将对移植免疫学有新的认识，发现新一代的生物标志物并确定下一代潜在的药物靶点。