Molecular Pathology of HIV/SIV Enteropathy

描述(申请人提供)：胃肠道(GI)是HIV/SIV感染和CD4+T细胞耗尽的主要目标。粘膜免疫系统的损害与各种胃肠道症状有关，统称为艾滋病肠病；通常以慢性腹泻和消瘦为特征。虽然我们对HIV/SIV肠病的认识最近有了很大的提高，但最近发现的microRNAs(MiRNAs)又增加了另一个新的复杂的基因表达调控因子，在这种疾病的分子发病机制中具有潜在的作用。MiRNAs是~21-23个核苷酸的非编码RNA，高度保守，通过靶向mRNAs的翻译抑制或降解来抑制基因表达。虽然miRNA的研究在各种类型的癌症中得到了广泛的报道，并且在心脏、神经、代谢和皮肤病中的发病率不断上升，但它们在艾滋病毒/SIV肠病等特发性胃肠道疾病中的作用尚不清楚，尚未得到解决。对患有慢性腹泻和消瘦的SIV感染猕猴的结肠组织进行了初步的基于锁定核酸的miRNA微阵列分析，发现几个miRNAs的表达显著放松。根据我们强有力的初步证据，我们假设，由于HIV/SIV感染，miRNA在胃肠道中的表达被解除调控，这有助于肠道结构和功能的破坏(艾滋病肠病)。这项研究的总体目标是1a。)确定响应SIV感染的胃肠道miRNA表达的全基因组时间变化(微阵列和miRNA克隆)，并确定是否可以识别免疫激活、炎症和上皮屏障破坏等致病事件的特异表达特征。)利用实时定量RT-PCR进一步证实所有差异表达的miRNAs。1C。)确定差异表达的miRNAs(原位杂交/免疫荧光)在结肠中的细胞定位，并验证在邻近的不同细胞类型之间是否存在表达差异。通过在肠道上皮和体外培养的原代肠道巨噬细胞中验证其假定的蛋白质靶点(质谱学)，确定炎症相关miR-212的功能意义。了解miRNAs及其假定的靶基因/蛋白的作用将为HIV/SIV肠病的发病机制提供重要的见解，并可能提供其他胃肠道炎症条件。通过这些研究发现的分子机制可能为艾滋病肠病等特发性胃肠道疾病的诊断和治疗开辟新的基于miRNA的策略。

DESCRIPTION (provided by applicant): The gastrointestinal (GI) tract is a major target of HIV/SIV infection and CD4+ T cell depletion. The damage to the mucosal immune system is associated with a variety of GI manifestations collectively called AIDS enteropathy; generally characterized by chronic diarrhea, and wasting. Although our understanding of HIV/SIV enteropathy has greatly improved lately, the recent discovery of microRNAs (miRNAs) has added yet another novel and complex regulator of gene expression with potential roles in the molecular pathogenesis of this disorder. miRNAs are ~21-23 nucleotide noncoding RNAs, highly conserved and suppress gene expression by targeting mRNAs for translational repression or degradation. While miRNA studies are being reported extensively in various types of cancer, and at increasing rates in cardiac, neurological, metabolic and skin diseases, their role in idiopathic GI disorders such as HIV/SIV enteropathy is unknown and yet to be addressed. Preliminary Locked Nucleic Acid-based miRNA microarray profiling of colon tissue from SIV-infected macaques with chronic diarrhea and wasting revealed significant deregulation in the expression of several miRNAs. Based on our strong preliminary evidence, we hypothesize that miRNA expression is deregulated in the GI tract consequent to HIV/SIV infection and that this contributes to disruption of enteric structure and function (AIDS enteropathy). The broad goals of this study are to 1a.) Identify genome wide temporal changes in miRNA expression (microarray and miRNA cloning) in the GI tract in response to SIV infection and determine if expression signatures specific to pathogenic events such as immune activation, inflammation, and epithelial barrier disruption can be identified 1b.) Further corroborate all miRNAs showing differential expression using real-time RT-PCR. 1c.) Determine cellular localization of differentially expressed miRNAs (in situ hybridization/immunofluorescence) in the colon and verify if differences in expression exist between cell types in close proximity 2.) Determine the functional significance of the inflammation associated miR-212 by validating its putative protein targets (mass spectrometry) in the intestinal epithelium and in in vitro cultured primary intestinal macrophages. Understanding the role of miRNAs and their putative target genes/proteins will provide important insights into the pathogenesis of HIV/SIV enteropathy and, possibly, other GI inflammatory conditions. Molecular mechanisms uncovered through these studies may open new miRNA-based strategies for the diagnosis and treatment of idiopathic GI disorders like AIDS enteropathy.