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）未转化为蛋白质，而是在调节转录方面起作用。有充分的证据表明，NCRNA是发育，细胞和免疫学过程中的关键参与者。一类NCRNA是1993年发现的microRNA（miRNA），据信它们几乎调节了所有人类基因的30％。每个miRNA都有几至200个靶基因，它们通过在转录水平（mRNA降解）或平移水平（导致蛋白质合成）抑制mRNA来调节。现在有超过670个已知的人类miRNA（Sanger mirbase），尽管只有少数人经过机械验证。当前的文献鉴定出与造血谱系细胞相关的30个miRNA，但仅在T和B细胞中记录了一个小子集。我们假设miRNA通过差异化和组成型表达的基因来调节T和B淋巴细胞激活，分化和存活，在调节人类免疫力中起关键作用。尽管许多研究描述了与免疫相关的转录本和蛋白质策划移植免疫反应的研究，但对于调节这种基因组复杂性知之甚少。该建议的前提是，在这种复杂性中识别miRNA调节的分子网络将以无法通过简单的基因表达分析或蛋白质组学来实现的方式来阐明关键途径。为了在我们的初步研究中测试这一前提，我们使用了整个外显子阵列进行mRNA和一种新型的STEM-LOOP QPCR方法，用于420个已知的miRNA来研究人T和B细胞活化。结果表明，62个差异表达的miRNA，其中42个以前没有与淋巴细胞或造血谱系调节相关，超过3,000多个差异表达的mRNA，独特的miRNA谱，区分T和B细胞激活，以及许多高值候选的高值候选物，用于生物学上有明显的分子分子构成的miRNA调节。途径。该建议的目的是完成对人T和B细胞激活中miRNA表达的完整分析，以研究新的可能性，即在细胞核中miRNA的转录后处理后的新可能性很重要，使用串联质谱蛋白组和下一步生成深层测序（Roche 454 FLX）在激活过程中发现蛋白质，mRNA和miRNA表达与分子网络调节之间的相关性，并在生物学上具有重要意义的途径中验证许多高值miRNA候选者。如果我们的多维“ OMICS”方法成功，那么它是一个概念证明，努力组织一个大型协作小组的全部力量，以验证每种细胞类型的所有主要途径，从而确定免疫抑制的影响，将结果与结果相关联，将结果与结果相关联，将结果与之相关。来自移植患者的临床样本，旨在促进我们从单个基因连接机制水平到综合分子网络的水平的移植免疫学的理解。 公共卫生相关性：microRNA是通过抑制蛋白质的合成来起作用的一类令人兴奋的细胞过程调节剂。我们的初步结果表明，在免疫激活过程中，超过60个microRNA参与了人类淋巴细胞的激活。本工作的目的是应用多维系统生物学或“ OMIC”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。面临的挑战是制定和验证可以降低基因表达和蛋白质组学数据当前复杂性的新策略，回到离散的分子网络，并在基于假设驱动的基于机制的实验中验证这些策略。完成这种进化后，我们将对移植免疫学有新的了解，发现新一代的生物标志物并确定下一代潜在的药物靶标。