MicroRNA-9 controls the expression of Granuphilin/Slp4 and the secretory response of insulin-producing cells
MicroRNA-9 controls the expression of Granuphilin/Slp4 and the secretory response of insulin-producing cells
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DOI：
10.1074/jbc.m601225200
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发表时间：
2006-09-15
影响因子：
4.8
通讯作者：
Regazzi, Romano
中科院分区：
文献类型：
--
作者：
Plaisance, Valerie;Abderrahmani, Amar;Regazzi, Romano
胰岛β细胞的胰岛素释放对血糖的稳态起着至关重要的作用。已经确定了几种控制胰岛素胞吐的蛋白质，但决定β细胞分泌机制组件表达的因素在很大程度上仍不清楚。MicroRNAs是新近发现的小分子非编码RNA，可作为基因表达的抑制因子。我们发现，在胰岛素分泌细胞中过表达mir-9会减少葡萄糖或钾引起的胞吐作用。我们发现mir-9通过减少转录因子ONECUT-2的表达，进而通过增加Granuphin/Slp4的水平来发挥作用，Granuphin/Slp4是一种与β细胞分泌颗粒相关的Rab GTPase效应器，对胰岛素的释放起负面控制作用。事实上，电泳迁移率改变分析、染色质免疫沉淀和转染实验表明ONECUT-2能够与颗粒素启动子结合并抑制其转录活性。此外，我们还表明，通过RNA干扰沉默ONECUT-2增加了颗粒素的表达，并模拟了mir-9对刺激诱导的胞吐的影响。我们的数据提供了证据，证明在胰岛素产生细胞中，适当水平的mir-9对于维持适当的Granuphin水平和最佳的分泌能力是必需的。
Insulin release from pancreatic beta-cells plays an essential role in blood glucose homeostasis. Several proteins controlling insulin exocytosis have been identified, but the factors determining the expression of the components of the secretory machinery of beta-cells remain largely unknown. MicroRNAs are newly discovered small non-coding RNAs acting as repressors of gene expression. We found that overexpression of mir-9 in insulin-secreting cells causes a reduction in exocytosis elicited by glucose or potassium. We show that mir-9 acts by diminishing the expression of the transcription factor Onecut-2 and, in turn, by increasing the level of Granuphilin/Slp4, a Rab GTPase effector associated with beta-cell secretory granules that exerts a negative control on insulin release. Indeed, electrophoretic mobility shift assays, chromatin immunoprecipitation, and transfection experiments demonstrated that Onecut-2 is able to bind to the granuphilin promoter and to repress its transcriptional activity. Moreover, we show that silencing of Onecut-2 by RNA interference increases Granuphilin expression and mimics the effect of mir-9 on stimulus-induced exocytosis. Our data provide evidence that in insulin-producing cells adequate levels of mir-9 are mandatory for maintaining appropriate Granuphilin levels and optimal secretory capacity.