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

胰腺β细胞中环AMP对microRNA-375的调节

基本信息

  • 批准号:
    7880269
  • 负责人:
  • 金额:
    $ 20.3万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2010
  • 资助国家:
    美国
  • 起止时间:
    2010-07-01 至 2014-06-30
  • 项目状态:
    已结题

项目摘要

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.
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(如胰岛素分泌和能量稳态)的关键调节因子(Seino和Shibasaki,2005)。在胰腺中,当细胞暴露于激素、神经递质和其他营养促分泌素后,细胞内cAMP升高,并增强葡萄糖刺激的胰岛素分泌。因此,cAMP信号通路目前被2型糖尿病治疗药物靶向(Drucker and Nauck,2006)。环AMP还通过调节基因表达促进细胞功能、生长和存活的长期变化(Hinke等人,2004),并不是所有的都被完全理解。为了患者安全,重要的是要了解这些疗法的全方位靶点。在细胞生物学中,越来越多的人认识到称为microRNA的小的非编码RNA分子在发育和生理学中起着重要作用。微小RNA与靶基因杂交并通过RNA干扰(RNAi)机制抑制蛋白质合成(He和Hannon,2004; Meister和Tuschl,2004; Novina和Sharp,2004; Siomi和Siomi,2009)。然而,目前还不清楚microRNA是如何被cAMP调节的。该拟议项目的初步证据确定了cAMP的新靶点microRNA-375(miR-375),这是一种已知的胰岛素释放抑制剂。MiR-375是第一个显示在细胞中被cAMP靶向的微小RNA,并且是已知对cAMP有响应的第二个微小RNA(Vo et al.,2005年)。证据支持一种新的模型,即cAMP部分通过抑制这种microRNA来刺激葡萄糖刺激的胰岛素分泌。该提案验证了miR-375在转录上被细胞中的cAMP信号抑制并且该信号通过蛋白激酶A(PKA)途径发生的假设。特异性目的1测试了miR-375响应于cAMP而被转录抑制而非转录后降解的模型。两项实验将用于检测转录抑制、核连续试验和染色质免疫沉淀,一项实验将用于检测转录后降解、脉冲追踪RNA标记。如果确定机制为转录抑制,则将使用荧光素酶报告基因测定来鉴定介导抑制的DNA启动子序列。Specific Aim 2测试了miR-375及其基因靶点受PKA通路调节的模型,PKA通路是一种由cAMP激活的关键蛋白激酶。将使用PKA特异性抑制剂PKI在胰岛中进行实验,其将测试miR-375抑制是否通过PKA介导。第二个实验将测试cAMP信号传导的生理激动剂是否抑制miR-375以及它们的作用是否是PKA依赖性的。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调节。该项目的成功完成将导致miR-375的cAMP调控模型,该模型可用于设计补充和增强靶向cAMP途径的现有治疗方法的糖尿病治疗方法。 公共卫生相关性:2型糖尿病病例数量的迅速增加给美国和世界的医疗保健系统造成了不可持续的负担,虽然这种疾病无法治愈,但有一些疗法可以减轻一些症状和并发症。几种治疗剂激活胰腺细胞中的信号通路,称为环磷酸腺苷(cAMP)第二信使级联反应,可促进餐后胰岛素分泌。该提案将表征cAMP信号传导的新靶点microRNA-375,其有可能用作补充疗法,以增强靶向cAMP信号传导途径的现有疗法的疗效。

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