MicroRNA regulation of human T and B cell activation

描述(申请人提供)：非编码RNA(NcRNAs)不被翻译成蛋白质，但具有转录调控功能。有强有力的证据表明，ncRNAs在发育、细胞和免疫过程中发挥着关键作用。1993年发现的一类ncRNAs是microRNAs(MiRNAs)，据信它调控着几乎30%的人类基因。每个miRNA都有几个到多达200个靶基因，它们通过在转录水平(mRNA降解)或翻译水平(导致抑制蛋白质合成)抑制mRNA来调节这些基因。现在有超过670个已知的人类miRNAs(Sanger MiRBase)，尽管只有几个得到了机械验证。目前的文献发现了30个与造血系细胞相关的miRNA，但只有一小部分记录在T和B细胞中。我们假设，miRNAs通过靶向调节T和B淋巴细胞激活、分化和存活的关键差异和结构性表达基因，在调节人类免疫功能中发挥关键作用。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。这一建议的前提是，在这种复杂性中识别miRNA调控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验证这一前提，我们使用了完整的外显子mRNA阵列和一种新的针对420个已知miRNAs的茎环qPCR方法来研究人类T和B细胞的激活。结果发现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，3000多个差异表达的mRNAs，独特的区分T和B细胞激活的miRNA图谱，以及一些高价值的miRNA调控生物学意义的分子途径。这项建议的目的是完成对人类T和B细胞激活过程中miRNA表达的全面分析，探索细胞核中miRNA前体转录后处理的新可能性，利用串联质谱仪蛋白质组学和下一代深度测序(Roche 454Flx)来发现蛋白质、mRNA和miRNA表达与激活过程中分子网络调控之间的关系，并验证一些在生物学意义上具有重要意义的途径中的高价值miRNA候选基因。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，以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相关联，并旨在将我们对移植免疫学的理解从单个基因连锁机制的水平推进到集成分子网络。 与公共健康相关：microRNAs是一类潜在的令人兴奋的细胞过程调节因子，它通过抑制蛋白质的合成发挥作用。我们的初步结果表明，在免疫激活过程中，超过60个microRNAs参与了人淋巴细胞的激活。本工作的目的是应用多维系统生物学或“组学”方法，包括基因图谱、蛋白质组学和下一代测序，以了解microRNAs在人类免疫反应中调控的机制和途径。挑战是开发和验证新的策略，这些策略可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，并在假设驱动的、基于机制的实验中验证这些策略。当这一进化完成时，我们将对移植免疫学有一个新的理解，发现新一代生物标记物，并确定下一代潜在的药物靶点。

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.