ROLE OF MICRORNAS IN THE MOLECULAR PATHOGENESIS OF HIV/SIV ENTEROPATHY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 背景与目的：胃肠道是HIV/SIV感染的主要靶点。虽然我们对HIV/SIV肠病的认识有了很大的提高，但最近发现的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。MicroRNAs(MiRNAs)是由基因组转录的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。我们使用SIV感染的恒河猴模型研究了11个miRNAs在胃肠道疾病和炎症中的作用。方法：收集10只SIV感染的慢性腹泻猕猴(G1)和5只未感染的对照猕猴(G2)尸检时的结肠组织。应用QRT-PCR、原位杂交/免疫荧光、组织病理学和质谱学等方法研究了miR-21、miR-125b、miR-132、miR-142-3p、miR-142-5p、miR-146a、miR-155、miR-203、miR-212、miR-223和miR-338等与炎症相关的基因的作用。结果：G1代猕猴均有慢性腹泻、消瘦、结肠炎等症状。与G2组相比，G1组猕猴结肠中miR-142-3p、miR-142-5p(5-6倍)、miR-212(3倍)、miR-21(2倍)的表达显著增加。有趣的是，miR-125b和miR-203在G1动物中表达下调(2-3倍)。两组间miR-132、miR-146a、miR-155、miR-223和miR-338的表达无明显差异。用锁定核酸修饰的miR-212探针进行原位杂交，发现结肠上皮和固有层细胞(巨噬细胞)有胞浆和胞核的定位。在体外培养的肠巨噬细胞中过表达miR-142-3p和miR-212两个miRNAs后，我们用质谱仪验证了这两个miRNAs的几个预测的信使核糖核酸靶点。结论：HIV/SIV感染引起的miRNAs细胞/组织特异性表达的失控可能会破坏肠道上皮和粘膜免疫系统之间的功能关系，导致肠道结构和功能的改变。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Background & Aims: The Gastrointestinal tract (GIT) is a major target of HIV/SIV infection. Although our understanding of HIV/SIV enteropathy has greatly improved, the recent discovery of miRNAs has added yet another novel and complex regulator of gene expression with potential roles in the molecular pathogenesis of this disorder. microRNAs (miRNAs) are genomically transcribed, ~21-23 nucleotide noncoding RNAs that are highly conserved and suppress gene expression by targeting mRNAs for translational repression or degradation. We investigated the contribution of 11 miRNAs to GIT disease and inflammation using the SIV-infected rhesus macaque model. Methods: Colon tissue was collected at necropsy from 10 SIV-infected (G1) with chronic diarrhea and 5 uninfected control macaques (G2). The contributions of miR-21, miR-125b, miR-132, miR-142-3p, miR-142-5p, miR-146a, miR-155, miR-203, miR-212, miR-223 and miR-338, previously known to be associated with inflammation were investigated using QRT-PCR, In situ hybridization/Immunofluorescence, histopathology and mass spectrometry. Results: All G1 macaques had chronic diarrhea, wasting and colitis. Significant to moderate increase in the expression of miR-142-3p, miR-142-5p (5-6-fold), miR-212 (3-fold), miR-21 (2-fold) was observed in the colon of G1 macaques compared to G2 animals. Interestingly, miR-125b and miR-203 were downregulated (2-3 fold) in G1 animals. No change in miR-132, miR-146a, miR-155 , miR-223 and miR-338 expression was observed between the groups. In situ hybridization using Locked-nucleic acid modified miR-212 probes revealed cytoplasmic and nuclear localization in the colonic epithelium and lamina propria cells (macrophages). Using mass spectrometry, we validated several predicted mRNA targets of miR-142-3p and miR-212 following over expression of both miRNAs in in vitro cultured primary intestinal macrophages. Conclusion: Our findings suggest that deregulation in cell/tissue-specific expression of miRNAs occurring in response to HIV/SIV infection may disrupt the functional relationship between the intestinal epithelium and the mucosal immune system causing alterations in intestinal structure and function.