CaMK Ⅱ磷酸化介导STAT5基因沉默诱发的促MIN6细胞胰岛素分泌

Silencing of Stat5 Promotes Insulin Secretion in Mouse MIN6 Cells via Phosphorylation of CaMK Ⅱ

信号转导及转录活化蛋白5(signal transducer and activator of transcription 5,STAT5)在乳腺发育、免疫应答、细胞代谢、造血及肿瘤的发生发展中发挥重要作用,但对胰岛细胞功能影响研究甚少。本研究旨在探讨STAT5对小鼠胰岛肿瘤MIN6细胞胰岛素分泌的影响及作用机制。电穿孔法转染小干扰RNA(siRNA)敲减STAT5基因表达后,实时定量PCR和蛋白质印迹法显示,与对照干扰RNA转染的细胞比较,Stat5 siRNA转染细胞的mRNA及蛋白质表达水平分别约70%和67%(P<0.01),提示成功构建了Stat5基因沉默模型。酶联免疫吸附法结合蛋白质印迹法显示,与对照组细胞比较,Stat5 siRNA转染细胞在不同浓度葡萄糖刺激下,胰岛素分泌能力提高大约1倍(P<0.05);同时,钙调蛋白依赖的蛋白激酶Ⅱ(calmodulin-dependent protein kinaseⅡ,Ca MKⅡ)的磷酸化水平亦随之加强。加入Ca MKⅡ抑制剂AIPⅡ(auto camtide-2 related inhibitory peptideⅡ)可明显抑制Stat5 siRNA转染导致的细胞胰岛素分泌水平增加(P<0.01)。上述结果表明,沉默Stat5基因后可通过增强Ca MKⅡ的磷酸化促进MIN6细胞胰岛素的分泌。

Signal transducer and activator of transcription 5（ STAT5） has been shown to play important roles in mammary gland development,immune response,cell metabolism,and hematopoiesis,as well as in tumor development and progression. However,less is known about the function of STAT5 in islet cells. This study aimed to investigate the effect and mechanism of STAT5 on insulin secretion in mouse MIN6 tumor cells. Real time-quantitative PCR（ RT-q PCR） and Western blot showed that the mRNA and protein expression of Stat5 was decreased to in the 70% and 67% respectively（ P 0. 01） by RNAi,comparing to electroporation-mediated transfection of a scrambled RNA. ELISA and Western blotting showed that insulin secretion in the Stat5 siRNA-transfected cells were increased about 1-fold with glucose stimulation of different concentrations for 48 hours（ P 0. 05）. The phosphorylation of calmodulindependent protein kinase Ⅱ（ Ca MK Ⅱ） were increased. Auto camtide-2 related inhibitory peptide Ⅱ（ AIPⅡ）,a Ca MKⅡ inhibitor,inhibited the insulin secretion in Stat5 silenced cells（ P 0. 01）. These data suggested that silencing of Stat5 promoted insulin secretion in MIN6 cells by increasing Ca MK Ⅱphosphorylation.