Wang Guiliang, Jiang Lei, Song Juan, Zhou Shu-Feng, Zhang Huali, Wang Kangkai, Xiao Xianzhong
Laboratory of Shock, Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha 410008, Hunan, China.
Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL 33612, USA.
Int J Mol Sci. 2014 Oct 10;15(10):18206-20. doi: 10.3390/ijms151018206.
Mipu1 (myocardial ischemic preconditioning upregulated protein 1), a novel rat gene recently identified in our lab, was expressed abundantly and predominantly in the brain and heart and upregulated in myocardium during myocardial ischemia/reperfusion in rats. In our previous study we found that Mipu1 was an evolutionarily conserved zinc finger-containing transcription factor. However, whether Mipu1 confers myocardial protection remains unknown. In this study, H9c2 myogenic cells were treated with hydrogen peroxide (H2O2) to simulate oxidative stress during myocardial ischemia-reperfusion injury. The expression of Mipu1 at mRNA and protein levels was detected by RT-PCR and Western blotting analysis. To study the effect of Mipu1 on apoptosis and expression of Fas induced by H2O2, full-length Mipu1 cDNA and Mipu1-RNAi plasmids were transiently transfected into H9c2 myogenic cells, and flow cytometry was used to quantitate the percentage of apoptotic cells. The expression of Fas was analyzed by Western blotting assay. The DNA binding and transcription activities of Mipu1 to the Fas promoter were detected by chromatin immunoprecipitation and luciferase reporter assays. The results showed that exposure of H9c2 myogenic cells to H2O2 resulted in a dose- and time-dependent increase in Mipu1 mRNA and protein levels; Mipu1 over-expression inhibited H2O2-induced apoptosis and upregulation of Fas induced by H2O2 in H9c2 myogenic cells; and knockdown of Mipu1 by RNAi promoted apoptosis and upregulation of Fas induced by H2O2. The chromatin immunoprecipition and reporter assays showed the DNA binding and transcription suppressor activities of Mipu1 to Fas promoter region. These results indicate that Mipu1 protected H9c2 myogenic cells from H2O2-induced apoptosis through inhibiting the expression of Fas.
Mipu1（心肌缺血预处理上调蛋白1）是我们实验室最近鉴定出的一个新的大鼠基因，在大鼠脑和心脏中大量且主要表达，在心肌缺血/再灌注期间心肌中表达上调。在我们之前的研究中，我们发现Mipu1是一种进化上保守的含锌指转录因子。然而，Mipu1是否赋予心肌保护作用仍不清楚。在本研究中，用过氧化氢（H2O2）处理H9c2成肌细胞以模拟心肌缺血再灌注损伤期间的氧化应激。通过RT-PCR和蛋白质印迹分析检测Mipu1在mRNA和蛋白质水平的表达。为了研究Mipu1对H2O2诱导的凋亡和Fas表达的影响，将全长Mipu1 cDNA和Mipu1-RNAi质粒瞬时转染到H9c2成肌细胞中，并用流式细胞术定量凋亡细胞的百分比。通过蛋白质印迹分析检测Fas的表达。通过染色质免疫沉淀和荧光素酶报告基因检测来检测Mipu1与Fas启动子的DNA结合和转录活性。结果表明，H9c2成肌细胞暴露于H2O2导致Mipu1 mRNA和蛋白质水平呈剂量和时间依赖性增加；Mipu1过表达抑制H2O2诱导的H9c2成肌细胞凋亡和H2O2诱导的Fas上调；RNAi敲低Mipu1促进H2O2诱导的凋亡和Fas上调。染色质免疫沉淀和报告基因检测显示Mipu1对Fas启动子区域具有DNA结合和转录抑制活性。这些结果表明，Mipu1通过抑制Fas的表达保护H9c2成肌细胞免受H2O2诱导的凋亡。
