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，但在转录调节中起作用。有强有力的证据表明，ncRNA是发育，细胞和免疫过程中的关键参与者。一类ncRNA是1993年发现的microRNA（miRNAs），据信调节近30%的人类基因。每个miRNA具有几个到多达200个靶基因，它们通过在转录水平（mRNA降解）或翻译水平（导致蛋白质合成的抑制）抑制mRNA来调节靶基因。现在有超过670种已知的人类miRNAs（桑格miRBase），尽管只有少数被机械验证。目前的文献鉴定了30种与造血谱系细胞相关的miRNA，但在T和B细胞中仅记录了一小部分。我们推测miRNAs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性和组成性表达基因在调节人类免疫中发挥关键作用。尽管许多研究描述了免疫相关的转录本和蛋白质协调移植免疫反应，但对调控这种基因组复杂性知之甚少。这个提议的前提是，在这种复杂性中识别miRNA调节的分子网络将以一种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mRNA的全外显子阵列和420种已知miRNA的新型茎环qPCR方法来研究人T和B细胞活化。结果显示62个差异表达的miRNA，其中42个先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细胞活化的独特miRNA谱以及许多生物学上重要的分子途径的miRNA调节的高价值候选物。本提案的目的是完成对人类T和B细胞活化中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的新可能性，使用串联质谱蛋白质组学和下一代深度测序。（Roche 454 FLX）来发现激活过程中蛋白质、mRNA和miRNA表达与分子网络调节之间的相关性，并在生物学意义的途径中验证许多高价值的miRNA候选物。如果我们的多维“组学”方法是成功的，那么它是一个概念的证明，努力组织一个大型合作小组的全部力量，验证每种细胞类型的所有主要途径，确定免疫抑制的影响，将结果与来自移植患者的临床样本相关联，旨在从个体基因水平促进我们对移植免疫学的理解，连接机制到整合的分子网络。 公共卫生相关性：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来发挥作用。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。本工作的目的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，以了解人体免疫反应中microRNA调控的机制和途径。挑战在于开发和验证新的策略，这些策略可以降低基因表达和蛋白质组数据的当前复杂性，回到离散的分子网络，并在假设驱动的、基于机制的实验中验证这些策略。当这种进化完成后，我们将对移植免疫学有新的认识，发现新一代生物标志物并识别下一代潜在的药物靶点。