Molecular Pathology of HIV/SIV Enteropathy

描述（由申请方提供）：胃肠道（GI）是HIV/SIV感染和CD 4 + T细胞耗竭的主要靶点。对粘膜免疫系统的损害与统称为AIDS肠病的各种GI表现有关;通常以慢性腹泻和消瘦为特征。虽然我们对HIV/SIV肠病的理解最近有了很大的提高，但最近发现的microRNAs（miRNAs）又增加了另一种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。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，并且在心脏病、神经病、代谢病和皮肤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，有待解决。初步的基于锁核酸的miRNA微阵列分析来自SIV感染的患有慢性腹泻和消瘦的猕猴的结肠组织，揭示了几种miRNA表达的显著失调。基于我们强有力的初步证据，我们假设HIV/SIV感染后胃肠道中的miRNA表达失调，这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1a）。鉴定响应SIV感染的胃肠道中miRNA表达的全基因组时间变化（微阵列和miRNA克隆），并确定是否可以鉴定特异于致病事件（如免疫激活、炎症和上皮屏障破坏）的表达特征1b）。使用实时RT-PCR进一步证实所有显示差异表达的miRNA。1c.）确定差异表达的miRNA在结肠中的细胞定位（原位杂交/免疫荧光），并验证在非常接近的细胞类型之间是否存在表达差异2。通过在肠上皮和体外培养的原代肠巨噬细胞中验证其推定的蛋白质靶点（质谱法），确定炎症相关miR-212的功能意义。了解miRNAs的作用及其推定的靶基因/蛋白质将为HIV/SIV肠病的发病机制以及可能的其他GI炎症状况提供重要的见解。通过这些研究发现的分子机制可能为诊断和治疗特发性胃肠道疾病（如艾滋病肠病）开辟新的基于miRNA的策略。 公共卫生相关性：这项拟议的研究将首次调查小调节核糖核酸（microRNA）在HIV/SIV肠病发病机制中的作用。HIV/SIV肠病是一种特发性疾病，影响三分之二的HIV感染患者，确切原因仍不清楚。这类研究将进一步增强我们对该疾病分子发病机制的理解，并有助于在未来开发更好的诊断和治疗策略。

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. PUBLIC HEALTH RELEVANCE: The proposed study for the first time will investigate the role of small regulatory ribonucleic acids (microRNAs) in the pathogenesis of HIV/SIV enteropathy. HIV/SIV enteropathy is an idiopathic disorder that affects two-thirds of HIV infected patients and the exact causes still remain obscure. Studies of this kind will further enhance our understanding of the molecular pathogenesis of this order and will help develop better diagnostic and therapeutic strategies in the future.