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

描述（由申请人提供）：环磷酸腺苷（cAMP）第二信使信号级联是多种细胞过程的关键调节器，如胰岛素分泌和能量稳态（Seino和Shibasaki， 2005）。在胰腺细胞中，细胞内cAMP在暴露于激素、神经递质和其他营养分泌物后升高，并增强葡萄糖刺激的胰岛素分泌。因此，cAMP信号通路目前正成为2型糖尿病治疗的靶点（Drucker和Nauck， 2006）。Cyclic-AMP还通过调控基因表达促进细胞功能、生长和存活的长期变化（Hinke et al., 2004），但并非所有这些都被完全了解。为了患者安全，了解这些治疗方法的全部靶点是很重要的。在细胞生物学中，越来越多的人意识到小的非编码RNA分子（称为microrna）在发育和生理中起着重要作用。MicroRNAs与靶基因杂交并通过RNA干扰（RNAi）机制抑制蛋白质合成（He and Hannon, 2004; Meister and Tuschl, 2004; Novina and Sharp, 2004; Siomi and Siomi, 2009）。然而，目前尚不清楚细胞中cAMP是如何调控microrna的。这个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是细胞中第一个被cAMP靶向的microRNA，也是第二个已知对cAMP有反应的microRNA （Vo et al., 2005）。这些证据支持一种新的模型，即cAMP通过抑制这种微小rna来刺激葡萄糖刺激的胰岛素分泌。该提议验证了miR-375在细胞中受到cAMP信号的转录抑制，并且这种信号通过蛋白激酶A （PKA）途径发生的假设。特异性Aim 1测试了miR-375在cAMP作用下被转录抑制而非转录后降解的模型。两个实验将用于测试转录抑制，核运行试验和染色质免疫沉淀，一个实验将用于测试转录后降解，脉冲追踪RNA标记。如果机制被确定为转录抑制，那么荧光素酶报告基因检测将用于鉴定介导这种抑制的DNA启动子序列。特异性Aim 2验证了miR-375及其基因靶点受PKA通路调控的模型，PKA通路是cAMP激活的关键蛋白激酶。我们将在胰岛中使用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结合位点被cAMP调控。该项目的成功完成将产生cAMP调控miR-375的模型，该模型可用于设计糖尿病治疗方法，以补充和增强针对cAMP途径的现有治疗方法。

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.