Regulation of microRNA-375 by cyclic-AMP in pancreatic beta-cells

描述(申请人提供)：环磷酸腺苷(CAMP)第二信使信号级联是胰岛素分泌和能量平衡等多种细胞过程的关键调节因子(Seino和Shibasaki，2005)。在胰腺细胞中，暴露于激素、神经递质和其他营养促分泌剂后，细胞内cAMP升高，并增强葡萄糖刺激的胰岛素分泌。出于这个原因，cAMP信号通路目前是2型糖尿病治疗药物的靶点(Drucker和Nauck，2006)。环磷酸腺苷还通过调节基因表达来促进细胞功能、生长和存活的长期变化(Hinke等人，2004年)，但并不是所有的都被完全理解。为了患者的安全，了解这些疗法靶点的全谱是很重要的。细胞生物学中越来越多的人意识到，被称为microRNAs的非编码小RNA分子在发育和生理中发挥着重要作用。MicroRNAs通过RNA干扰(RNAi)机制与目标基因杂交并抑制蛋白质合成(他和Hannon，2004；Meister和Tuschl，2004；Novina和Sharp，2004；siomi和siomi，2009)。然而，目前还不清楚cAMP是如何在细胞内调节microRNAs的。这个拟议项目的初步证据确定了cAMP的一个新靶点，microRNA-375(miR-375)，一种已知的胰岛素释放抑制剂。MIR-375是第一个被cAMP-细胞靶向的microRNA，也是第二个已知对cAMP有反应的microRNA(Vo等人，2005年)。证据支持一种新的模型，即cAMP刺激葡萄糖刺激的胰岛素分泌，部分是通过抑制这种微小RNA。这一建议验证了一种假设，即miR-375在转录上受到细胞内cAMP信号的抑制，并且这种信号通过蛋白激酶A(PKA)途径发生。特定目标1测试了miR-375在转录上被抑制，而不是转录后降解的模型，以回应cAMP。两个实验将被用来测试转录抑制、核运行试验和染色质免疫沉淀，一个实验将被用来测试转录后降解、脉冲追逐RNA标记。如果机制被确定为转录抑制，那么荧光素酶报告分析将被用于鉴定介导抑制的DNA启动子序列(S)。特异性目标2测试了miR-375及其基因靶点受PKA途径调控的模型，PKA通路是一种由cAMP激活的关键蛋白激酶。将在胰岛中使用PKA特异性抑制剂PKI进行实验，以测试miR-375的抑制是否通过PKA介导。第二个实验将测试cAMP信号的生理激动剂是否抑制miR-375，以及它们的作用是否依赖于PKA。第三个实验将测试miR-375靶基因是否通过其miR-375结合位点受到cAMP的调控。该项目的成功完成将导致一个针对miR-375的cAMP调节模型，该模型可用于设计补充和增强针对cAMP途径的现有疗法的糖尿病疗法。 与公共卫生相关：2型糖尿病病例数量的快速上升正在给美国和世界的医疗保健系统造成不可持续的负担，虽然这种疾病没有治愈的方法，但有一些疗法可以缓解一些症状和并发症。几种疗法激活了胰腺细胞中的一条信号通路，称为环磷酸腺苷(CAMP)第二信使级联，它促进了餐后胰岛素的分泌。这项提议将表征cAMP信号通路的一个新靶点，microRNA-375，它有可能被用作补充疗法，以增强针对cAMP信号通路的现有疗法的疗效。

DESCRIPTION (provided by applicant): The cyclic-adenosine monophosphate (cAMP) second messenger signaling cascade is a key regulator of diverse cellular process such as insulin secretion and energy homeostasis (Seino and Shibasaki, 2005). In pancreatic ¿-cells, intracellular cAMP is elevated following exposure to hormones, neurotransmitters, and other nutrient secretagogues, and potentiates glucose-stimulated insulin secretion. For this reason, the cAMP signaling pathway is currently being targeted by therapeutics for type 2 diabetes mellitus (Drucker and Nauck, 2006). Cyclic-AMP also promotes long-term changes in ¿-cell function, growth, and survival through regulation of gene expression (Hinke et al., 2004), not all of which are completely understood. For patient safety it is important to understand the full-spectrum of targets of these therapeutics. There is a growing awareness in ¿- cell biology that small non-coding RNA molecules called microRNAs play a major role in development and physiology. MicroRNAs hybridize with target genes and repress protein synthesis via an RNA interference (RNAi) mechanism (He and Hannon, 2004; Meister and Tuschl, 2004; Novina and Sharp, 2004; Siomi and Siomi, 2009). It is unclear, however, how microRNAs are regulated by cAMP in ¿-cells. Preliminary evidence for this proposed project identifies a new target of cAMP, microRNA-375 (miR-375), a known inhibitor of insulin release. MiR-375 is the first microRNA shown to be targeted by cAMP in ¿-cells and only the second microRNA known to be responsive to cAMP (Vo et al., 2005). The evidence supports a new model whereby cAMP stimulates glucose-stimulated insulin secretion in part by repressing this microRNA. This proposal tests the hypothesis that miR-375 is transcriptionally repressed by cAMP signaling in ¿-cells and that this signaling occurs via the protein kinase A (PKA) pathway. Specific Aim 1 tests the model that miR-375 is transcriptionally repressed, and not post-transcriptionally degraded, in response to cAMP. Two experiments will be used to test for transcriptional repression, nuclear run-on assay and chromatin immunoprecipitation, and one experiment will be used to test for post-transcriptional degradation, pulse-chase RNA labeling. If the mechanism is determined to be transcriptional repression, then luciferase reporter assays will be used to identify a DNA promoter sequence(s) that mediates the repression. Specific Aim 2 tests the model that miR-375 and its gene targets are regulated by the PKA pathway, a key protein kinase that is activated by cAMP. An experiment will be conducted in pancreatic islets using the PKA-specific inhibitor, PKI, which will test whether miR-375 repression is mediated through PKA. A second experiment will test whether physiological agonists of cAMP signaling repress miR-375 and whether their effects are PKA-dependent. A third experiment will test whether miR-375 target genes are regulated by cAMP via their miR-375 binding site. Successful completion of this project should result in a model for cAMP regulation of miR-375 which could be used to design diabetes therapies that complement and enhance existing therapeutics that target the cAMP pathway. PUBLIC HEALTH RELEVANCE: The rapid rise in the numbers of cases of type 2 diabetes mellitus is creating an unsustainable burden on the health care systems of the U.S. and world, and while there is no cure for the disease, there are therapies which alleviate some of the symptoms and complications. Several therapeutics activate a signaling pathway in pancreatic ¿-cells called the cyclic-adenosine monophosphate (cAMP) second messenger cascade which boosts postprandial insulin secretion. This proposal will characterize a new target of cAMP signaling, microRNA-375, which has the potential to be used as a complementary therapy to enhance the efficacy of existing therapeutics that target the cAMP signaling pathway.