Molecular Pathology of HIV/SIV Enteropathy

描述（由申请人提供）：胃肠道是HIV/SIV感染和CD4+ T细胞耗竭的主要靶点。粘膜免疫系统的损害与多种胃肠道表现相关，统称艾滋病肠病；通常以慢性腹泻和消瘦为特征。尽管我们对HIV/SIV肠道病的理解最近有了很大的提高，但最近发现的microRNAs （miRNAs）又增加了一种新的复杂的基因表达调节因子，在这种疾病的分子发病机制中具有潜在的作用。mirna是约21-23个核苷酸的非编码rna，高度保守，通过靶向mrna进行翻译抑制或降解来抑制基因表达。虽然miRNA研究在各种类型的癌症中被广泛报道，并且在心脏、神经、代谢和皮肤疾病中的发病率越来越高，但它们在特发性胃肠道疾病（如HIV/SIV肠病）中的作用尚不清楚，也有待解决。对siv感染的慢性腹泻和消瘦猕猴结肠组织的初步锁定核酸miRNA微阵列分析显示，几种miRNA的表达明显失调。基于我们强有力的初步证据，我们假设HIV/SIV感染导致胃肠道中miRNA表达失调，从而导致肠道结构和功能的破坏（艾滋病肠病）。本研究的主要目标是：确定SIV感染后胃肠道miRNA表达（微阵列和miRNA克隆）在全基因组范围内的时间变化，并确定是否可以识别特异性致病事件（如免疫激活、炎症和上皮屏障破坏）的表达特征。使用实时RT-PCR进一步证实所有显示差异表达的mirna。1 c)。确定结肠中差异表达的mirna的细胞定位（原位杂交/免疫荧光），并验证在接近的细胞类型之间是否存在表达差异2。通过验证其在肠上皮和体外培养的原代肠巨噬细胞中推测的蛋白靶点（质谱法），确定炎症相关的miR-212的功能意义。了解mirna及其假定的靶基因/蛋白的作用将为了解HIV/SIV肠病以及可能的其他胃肠道炎症的发病机制提供重要的见解。通过这些研究揭示的分子机制可能为艾滋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.