Protective Effect Of MIPU1on Doxorubicin-induced Apoptosis In HEK293Cells

Objective:Doxorubicin is an anti-tumour chemical, however it causes a lot of side-effects, such as adriamycin cardiomyopathy and nephropathy and so on. It has been well documented that apoptosis contributes to its cytotoxicity. Mipul is a novel gene identified in our lab (GenBank accession number AY221750) which is up-regulated in myocardium of rat insulted with ischemic preconditionning. Recently, we founded that the function of Mipul as a transcriptional repressor with features of a KRAB type zinc finger protein. Bioinformatic analysis showed that Mipu1protein and its functinal structure are evolutionary conservation with high homology among human and rodent. Recently, we found that there have the core sequence ("CTTA") of Mipu1binding element within their promoter region of several apoptosis related genes,such as Bax, Bcl-2, Bid,P53and Bak etc. However, it is not clear whether MIPU1can attenuate the cytotoxicity of DOX by regulating these apoptosis-associated genes at the transcriptional level. So, in the present study, we projected to explore the inhibitory effects of MIPU1on DOX-mediated apoptosis and the mechanisms underlying. We hope that our studies would provide novel insight into the biological functions of Mipu1, and provide a new clue for preventing the side-effects of DOX during anticancer implication.Methods:Apoptosis was induced by Doxorubicin in this experiment. The eukaryotic expression plasmid pEGFP-MIPU1was constructed previously and delivered into the cells to increase the expression of MIPU1.There were four groups:pEGFP, pEGFP-MIPU1,pEGFP+DOX, pEGFP-MIPU1+DOX. Firstly, MTT assay was used to estimate the cells viability. Then apoptosis was assessed by using Hoechst staining, Caspase activity determination and flow cytometry (FCM). Thirdly, Western blot and RT-PCR were performed to detect the expression of Bax, P53and Bcl-2at the mRNA and protein levels respectively. Lastly, luciferase reporter gene assay was used to analyze the interation of MIPU1with Bax promoter. Results:(1) The cellular viability was decreased in HEK293cells treated with DOX (P<0.01VS pEGFP); After transfected with pEGFP-MIPU1, the overexpression of MIPU1significantly attenuated the mortality rate induced by DOX (P<0.05VS pEGFP+DOX).(2)The percentage of apoptotic cells was increased in DOX-induced HEK293cells (P<0.01VS pEGFP); while overexpression of MIPU1markedly decreased the cellular apoptosis treated with DOX (P<0.01VS pEGFP+DOX); The activity of Caspase-3was increased in HEK293cells treated with DOX (P<0.01VS pEGFP); After transfected with pEGFP-MIPU1, the activity of Caspase-3was decreased significantly (P<0.01VS pEGFP+DOX).(3) The overexpression of MIPU1reversed the decrease of Bcl-2expression in DOX-induced HEK293cells (P<0.05VS pEGFP+DOX).(4) The overexpression of MIPU1inhibited the increase of P53and Bax induced by DOX (P<0.05VS pEGFP+DOX). Meanwhile, the activity of the Bax promoter was also repressed by MIPU1(P<0.01VS pEGFP).Conclution:(1) MIPU1inhibited apoptosis in HEK293cells induced by DOX.(2) MIPU1promoted the expression of Bcl-2, and decreased the expression of Bax and P53in DOX-induced HEK293cells.(3) MIPU1possibly inhibited the activity of Bax promoter.