Effect Of HIF-1 On The Expression Of A Novel Gene Mipu1 In H9c2 Cardiomyocytes And Its Mechanism

Hypoxia-inducible factor-1(HIF-1) is the principal transcription factor involves in the regulation of gene expression in response to hypoxia.HIF-1 is a heterodimer comprised ofαandβsubunits,which are constitutively expressed.Theαsubunit is the activated and regulatory subunit of HIF-1.Some researches showed that up-regulated expression of HIF-1αby cobalt chloride(CoCl₂) treatment or transfecting HIF-1αgene confered cardioprotection against ischemic or ischemia/reperfusion (I/R) injury.More and more evidence suggested that HIF-1 was involved in cardioprotection induced by ischemic preconditioning(IP).The specific mechanism by which HIF-1 regulates cell survival in I/R is still unclear,but it likely involves multiple pathways that activate genes related to cardioprotection.Mipu1(myocardial ischemic preconditioning up- regulated protein 1),also known as Znf667,is a novel gene which was found to be up-regulated in rat heart after myocardial ischemic preconditioning in our laboratory.It served as a zinc finger nuclear transcription inhibitor.Bioinformatics analysis showed that a consensus hypoxia response element(HRE) of HIF-1 was found in the promoter of Mipu1.It indicated that HIF-1 may be the upstream regulator of Mipu1. This study was attempted to explore the effect of HIF-1 on the expression of the novel gene Mipu1 and its mechanism.In this study,The embryonic rat heart-derived cell line H9c2 cardiomyocytes were divided into five groups:(1) control group(Ctrl): H9c2 cells treated with PBS(Sphosphate buffer solution);(2) CoCl₂ group(Co):H9c2 cells treated with 200μmol/L CoCl₂ for 12h;(3) empty vector group(Neo):H9c2 cells transfected with pEF-BOS empty vector; (4) over-expressed HIF-1αgroup(HIF):H9c2 cells transfected with human HIF-1αfull-length plasmid(pEF-BOS-HIF-1α);(5) over -expressed HIF-1αplus CoCl₂ group(HIF+Co):H9c2 cells transfected with pEF-BOS-HIF-1αand treated with 200μmol/L CoCl₂ for 12h.Then reverse transcription-polymerase chain reaction(RT-PCR) was used to observe the expression of HIF-1αmRNA.Indirectly Immunofluorescence was used to observe the expression of HIF-1αprotien and its distribution in H9c2 cells.RT-PCR and Western Blot were used to observe the effect of HIF-1αon Mipu1 expression in H9c2 cells.Luciferase reporter gene was used to observe the effect of HIF-1αon the transcriptional activity of Mipu1 promoter.EMSA(Electrophoretic Mobility Shift Assays) and supershift was used to observe whether HIF-1 could bind to HRE of Mipu1 promoter.The results showed that:(1) The expression of H/F-1αmRNA did not change in CoCl₂ group (p＞0.05,vs Ctrl).However,it increased significantly in over-expressed HIF-1αgroup(p＜0.05 vs Neo).And the expression of HIF-1αmRNA did not change in over-expressed HIF-1αplus CoCl₂ group(p＞0.05 vs HIF).(2) HIF-1αprotein located in cytoplasm and nucleus of H9c2 cells in control group.The expression of HIF-1αprotein increased significantly and HIF-1αtranslocated from cytoplasm to nucleus in CoCl₂ group, over-expressed HIF-1αgroup and over-expressed HIF-1αplus CoCl₂ group.(3) The expression of Mipu1 mRNA and protein increased significantly in CoCl₂ group and over-expressed HIF-1αgroup(p＜0.05, Co vs Ctrl;p＜0.05,HIF vs Neo).However,it did not change in over-expressed HIF-1αplus CoCl₂ group(p＞0.05,vs HIF).(4) The transcriptional activity of Mipu1 promoter increased significantly in CoCl₂ group and over-expressed HIF-1αgroup(p＜0.01, Co vs Ctrl;p＜0.01,HIF vs Neo).However,it did not change in over-expressed HIF-1αplus CoCl₂ group(p＞0.05,vs HIF).(5) HIF-1 could bind to HRE of Mipu1 promoter.In conclusion,(1) HIF-1 promoted expression of Mipu1 in H9c2 cells through binding to HRE of Mipu1 promoter.(2) CoCl₂ promoted expression of Mipu1 in H9c2 cells through inducing expression of HIF-1αprotein.