Use of Micro-RNA Arrays to Identify MDSC Functional Pathways and Markers

描述(申请人提供)：髓系衍生抑制细胞(MDSC)是一种异质的、定义不清的免疫调节细胞群，在抑制肿瘤免疫和控制炎症过程中是一个关键的细胞群。目前，MDSC的功能是基于对包含抑制性和非抑制性细胞的表型定义的细胞群体的研究。没有明确定义功能MDSC的标记，也没有描述MDSC所特有的功能通路。我们最近发现，小鼠脾中的Gr1+CD11b+细胞是MDSC的前体细胞，并且活跃的调节MDSC只存在于肿瘤内或炎症部位。只有肿瘤或炎症来源的MDSC表达精氨酸酶I(Arg-I)和诱导型一氧化氮合酶(INOS)并抑制T细胞反应，明确地将功能性MDSC定位于活跃的炎症部位。因此，对来自脾和肿瘤的MDSC进行了阵列分析，以确定肿瘤来源的(功能性)MDSC的基因和微RNA(MiRNA)表达模式。基因阵列分析显示，与脾(前体)MDSC相比，肿瘤来源(功能性)MDSC中150个基因上调至少5倍，包括Arg-I(129倍)和iNOS(33倍)。到目前为止，在差异表达的新基因中，有两个基因，slc7a2和维生素D受体，被观察到调节MDSC T细胞抑制活性。因此，我们假设，识别与MDSC功能相关的miRNAs将识别新的靶点，从而能够识别和调节活跃的MDSC。此外，将miRNA分析与基因阵列数据相结合将增强功能MDSC特有的途径发现。差异miRNA阵列在相同的脾和肿瘤来源的MDSC上进行，用于基因阵列分析。MiRNA阵列显示有统计学意义的46个miRNAs上调2倍或更多，类似地40个miRNAs下调2倍或更多。我们建议利用差异miRNA阵列和基因阵列来识别与MDSC功能相关的新的MDSC特异基因和途径。调控miRNAs及其相关途径的鉴定将为更好地了解MDSC功能的分子基础奠定基础，也将为更好地靶向MDSC和/或鉴定活性MDSC的存在提供机会。提出了两个具体的目标：(1)识别与区分功能和前体MDSC显著相关的基因、基因组、通路和网络模块，以获得对生物学过程及其与癌症的相关性的全局洞察。假设：功能性MDSC利用不同于前体MDSC的不同功能通路来区分这两个群体。通过途径/网络建模和整合表达谱，可以在系统水平上检测到与功能相关的差异途径表达，以及(2)验证通过目标1中进行的分析确定的选定miRNA的生物学相关性。

DESCRIPTION (provided by applicant): Myeloid derived suppressor cells (MDSC) are a heterogeneous, ill-defined population of immune regulatory cells that are a critical cell population in suppressing tumor immunity and controlling inflammatory processes. Currently, MDSC function is based on studies of phenotypically defined cell populations that contain both suppressive and non-suppressive cells. Markers clearly defining functional MDSC are not available nor have functional pathways unique to MDSC been described. We recently showed that spleen Gr1+CD11b+ cells in mice, the prototypical MDSC phenotype, are precursor MDSC and that active regulatory MDSC are present only within a tumor or at the site of inflammation. Only tumor- or inflammation-derived MDSC to express Arginase I (Arg-I) and inducible nitric oxide synthase (iNOS) and suppress T cell responses, clearly localizing functional MDSC to active inflammatory sites. Thus, array analyses on MDSC both from spleen and tumor were performed to identify gene and micro-RNA (miRNA) expression patterns restricted to tumor-derived (functional) MDSC. Gene array analysis showed 150 genes to be upregulated at least 5-fold, including Arg-I (129-fold) and iNOS (33-fold), in tumor-derived (functional) MDSC compared to spleen (precursor) MDSC. Among the novel genes differentially expressed, to date two, slc7a2 and vitamin D receptor, were observed to regulate MDSC T cell suppressor activity. Thus we hypothesize that identifying miRNAs linked to MDSC function will identify new targets that will enable identification and modulation of active MDSC. Additionally, integrating the miRNA analysis with the gene array data will enhance pathway discovery unique to functional MDSC. Differential miRNA arrays were performed on the same spleen- and tumor-derived MDSC that were used for the gene array analysis. The miRNA arrays show a statistically significant 2-fold or greater up-regulation of 46 miRNAs and similarly a 2-fold or more down- regulation of 40 miRNAs. We propose to utilize the differential miRNA arrays in conjunction with the gene arrays to identify novel MDSC-specific genes and pathways that are associated with MDSC function. The identification of regulatory miRNAs and associated pathways will provide the foundation for better understanding the molecular basis for MDSC function and also will provide opportunity to better target MDSC and/or identify the presence of active MDSC. Two specific aims are proposed: (1) Identify genes, gene sets, pathways, and network modules that are significantly related to differentiating between functional and precursor MDSC to gain global insights of the biological process and its relevance to cancer. Hypothesis: functional MDSC utilize distinct functional pathways from precursor MDSC that will distinguish the two populations. The differential pathway expression linked to function can be detected at the systems level through pathway/network modeling and integrative expression profiling and (2) Validate the biological relevance of select miRNA identified via the analyses performed in Aim 1.