The Expression Pattern And Anti-apoptotic Effect Of Novel Gene Mipu1 In Cardiomyocytes

Myocardial ischemia is very common in clinic.It can be induced by many cardiovascular diseases such as coronary artherosclerosis and heart operation such as intracardiac surgery under extracorporal circulation and cardiac transplantation.A number of genes have been shown to be up-regulated during myocardial ischemia/reperfusion and playing important roles in myocardial protection,but there are clearly many missing elements to be found.Mipul is a novel gene cloned by Dr. Yuan in our laboratory which up-regulated in rat heart after transient myocardial ischemia-reperfusion,and further bioinformatics analysis indicated that Mipul gene was Composed of five exons and four introns with an open reading frame of 1827 bp and mapped to chromosome 1q12.1,encoding a polypeptide of 608 amino acids with an N-terminal KRAB domain and 14 C-terminal C2H2 zinc fingers.Dr.Jiang used Mipul fusion protein GST-Mipul to immobilize on glutathione-bound Sepharose beads to capture specific oligonucleotide sequences from a random oligonucleotide library and confirmed that Mipul could bind specifically to the consensus sequence 5′-TGTCTTATCGAA-3′,with TCTTA as the core sequence,exerting transcriptionally repressive effect on the promoter with its DNA binding sequence by binding to the promoters of downstream genes,which suggested that Mipul might act as a transcriptional repressor.In this study,the Mipul-6His fusion protein was purified by a His-bind resin and used to immunize New Zealand rabbits and produce a polycloned anti-Mipul serum.Using the anti-Mipul polyclonal antibody we found that H9c2 myogenic cells could express endogenous Mipul protein.We further observed the expression of Mipul in various tissues of rat and found that the highest levels of Mipul protein were detected in the brain and heart,moderate in the liver,kidneys,ovary and testis, and little in the lungs and spleen,which suggested that Mipul might play special functions in these tissues.Using fluorescent immunocytochemistry,we found that Mipul protein was localized in the nucleus of H9c2 myogenic cells.We further examined the expression and localization of Mipul in a rat myocardial ischemia model by using Northern blotting,real-time quantitative RT-PCR and Western blotting and found the the expression of Mipul was upregulated at 3h and lasted to 12h with a peak at 6h.Then we investigated the expression pattern of Mipul in a hydrogen peroxide(H₂O₂)-mediated oxidative stress/injury cell model in H9c2 cells by RT-PCR and Western blotting.The results showed that the expression of Mipul was up-regulated obviously in a time and dose-dependent manner after exposure to H₂O₂. The expression of Mipul mRNA and protein in H9c2 cells was increased significantly at the early phase(3h) and further increased up to the late phase(after 24 h) after H₂O₂ stimulation.Effect of Mipul overexpression and RNAi on H₂O₂-induced apoptosis was observed in H9c2 cells.Mipul was overexpressed by transfection of its full length cDNA plasmid and apoptosis H9c2 cells were characterized by flowcytometry.The result showed that overexpession of Mipul could reduce the percentage of apoptotic cells,and the down-regulation of Mipul by Mipul-RNAi could increase the percentage of apoptotic cells,suggesting that Mipul could protect H9c2 cells from apoptosis induced by H₂O₂.The effect of Mipul on the expression of apoptosis-associated gene Bax,Fas and Bcl-2 was observed in H9c2 cells.Western blotting was used to determined the effect of Mipul on the expression level of bax,fas and bcl-2 proteins in H9c2 cells. The results showed that the expression levels of Bax and fas protein were increased in H9c2 cells after being exposed to H₂O₂,but this up-regulation was alleviated by the overexpression of Mipul,and promoted by Mipul-RNAi;the results of luciferase reporter gene assay demonstrated that Mipul could inhibit the transcriptional activity of Bax and Fas.Chromatin immunoprecipitation showed the Mipul could bind to Bax and Fas promoter;The expression level of Bcl-2 protein was increased in H9c2 cells after being exposed to H₂O₂,and the up-regulation was also promoted by the overexpression of Mipul,and alliviated by Mipul-RNAi;The results of luciferase reporter gene assay demonstrated that Mipul could increase the transcriptional activity of Bcl-2.Chromatin immunoprecipitation showed that Mipul could not bind to Bcl-2 promoter. In a word,Mipul was localized in the nucleus of H9c2 cells;the expression of Mipul was abundant in the brain and cardiac tissues;the expression of Mipul was up-regulated in rat heart after myocardial ischemia and reperfusion and in H9c2 myogenic cells after exposure to H₂O₂;Mipul could inhibit apoptosis in H9c2 cells induced by H₂O₂,and its mechanism was associated with the suppression of pro-apoptotic gene Bax and Fas by direct binding to their promoters and the promotion of expression on anti-apoptotic gene Bcl-2 not by a direct transcriptional regulation.