Effect Of A Novel Gene Mipu1 On The Expression Of Bid Gene In H9c2 Cardiomyocytes And Its Mechanism

More and more evidence had suggested that the pathogenesis of many cardiovascular diseases was related to apoptosis in recent years. The apoptosis of myocardial cells could be induced by a variety of stimulus,such as hypoxia,ischemia-reperfusion,oxidative stress,tumor necrosis factorα(TNFα) and so on.TNFαmediated apoptosis by combining with TNF-R1 in myocardial cell membrane and activating the death receptor pathway.Mipu1 is a noval myocardial ischemic preconditioning up-regulated gene cloned by our laboratory(GenBank accession number AY221750),and it has been confirmed that Mipu1 is a zinc finger protein and a transcriptional factor.Because bioinformatics analysis showed that the promoter region of Bid(a Bcl-2 family member) contains the binding sites of Mipu1,so we want to observe the effect of Mipu1 on TNFα-induced apoptosis of H9c2 cardiomyocytes,and to explore the regulation of Mipu1 upon apoptosis-related gene Bid.Apoptosis was induced by exposure of H9c2 cells to TNFαwith the final concentration of 20 ng/ml.H9c2 cells overexpressed Mipu1 by transfecting transiently with eukaryotic expression plasmid pcDNA3.1-Mipu1.There were four groups:pcDNA3.1,pcDNA3.1+TNFα, pcDNA3.1-Mipu1,pcDNA3.1-Mipu1+TNFα.In order to observe the effect of Mipu1 on TNFα-induced apoptosis,the percentage of apoptotic cells and activity of Caspase-8,9,3 were measured by Hoechst staining,flow cytometry(FCM),and Caspase activity detection;In order to observe effect of Mipu1 on the expression of Bid,protein and mRNA of Bid were measured by indirect immunofluorescence assay, Western-blot,RT-PCR and Real-time quantitative PCR;In order to explore furtherly the transcriptional regulation of Mipu1 upon Bid gene, luciferase reporter gene and Target Detection Assay were used.The main findings were as follows:①The percentage of apoptotic cells was increased in H9c2 cells treated with TNFα(p＜0.01 VS pcDNA3.1);After transfected with pcDNA3.1-Mipu1,the increase of apoptotic cells percentage induced by TNFαwas inhibited significantly(p＜0.01 VS pcDNA3.1+TNFα).The activities of Caspase-8,Caspase-3 were increased in H9c2 cells treated with TNFα(p＜0.01 VS pcDNA3.1); After transfected with pcDNA3.1-Mipu1,the increase of the activities of Caspase-8,Caspase-3 induced by TNFαwas inhibited significantly(p＜0.01 VS pcDNA3.1+TNFα);But the activity of Caspase-9 had no significant difference in four groups.②Indirect immunofluorescence assay demonstrated that Bid located in cytoplasm and the expression of Bid protein was reduced in H9c2 cells transfected with pcDNA3.1-Mipu1 compared with H9c2 cells transfected with pcDNA3.1.The expression of Bid protein and mRNA were down-regulated in H9c2 cells transfected with Mipu1(p＜0.05 VS pcDNA3.1);After treated with TNFα,the expression of Bid protein and mRNA were increased(p＜0.05 VS pcDNA3.1),but this increase was alleviated by overexpression of Mipu1 (p＜0.05 VS pcDNA3.1+TNFα);Further more,Western-blot showed that the expression of Bid protein gradually decreased with the increase of pcDNA3.1-Mipu1 transfected to H9c2 cells.③Transcriptional activity of Bid gene was down-regulated in H9c2 cells transfected with pcDNA3.1-Mipu1(p＜0.05 VS pcDNA3.1);After treated with TNFα, transcriptional activity of Bid gene was significantly increased(p＜0.01 VS pcDNA3.1),but this increase was significantly alleviated by overexpression of Mipu1(p＜0.01 VS pcDNA3.1+TNFα);Moreover, the transcriptional activity of Bid gene gradually decreased with the increase of pcDNA3.1-Mipu1 transfected to H9c2 cells.Target Detection Assay showed that Mipu1 could bind the promoter region of Bid gene in vitro.Then it was concluded that:①Mipu1 overexpression could inhibit TNFα-induced apoptosis of H9c2 cells,and its mechanism might be related to Mipu1 inhibiting the activities of Caspase-8 and Caspase-3.②Mipu1 could inhibit the expression of Bid gene,and its mechanism might be related to Mipu1 down-regulating the transcription of Bid gene by directly binding to the promoter region of Bid gene.