Study On The Transcriptional Regulation Mechanism Of Prestin Gene In HEI-OC1 Cells Upon Oxidative Stress

Objective Noise-induced hearing loss(NIHL)is one of the most common occupational disease,high level of reactive oxygen species(ROS)produced by oxidative stress is not only the important mechanism of NIHL cochlear hair cell damage,but also the pathologic basis of various sensory hearing loss.Prestin protein is an important acoustic functional protein only expressing in the cochlear outer hair cells,it is the molecular basis of electromotility and cochlear amplification of mammalian hair cell.The research is amied to provide theoretical basis for preventing from NIHL by exploring the alteration of Prestin in HEI-OC1(House Ear Institute-outraged of Corti 1)cell in oxidative stress state and the molecular cechanism of Prestin gene transcriptional regulation under oxidative stress.Methods HEI-OC1 cells were incubated with four different concentrations(0 ?M,50 ?M,100 ?M,and 200 ?M)of tert-butyl hydroperoxide(t-BHP)for 24 or 48 h to construct oxidative stress damage model.q RT-PCR and Western Blot were used to detect the expression level of Prestin and AP-2? at m RNA and protein level in HEI-OC1 cells in oxidative stress state,it mainly amied to observe the influence of oxidative damage.Reverse Ch IP and LC-MS assays were performed to identify proteins that could bind to promoter region of Prestin gene,followed by preliminary screening for transcription factors(TF)that can modulate Prestin gene expression under the condition of oxidative stress.According to the expression of TFs m RNA under the oxidative stress,the most vital TF participating in the transcriptional regulation of Prestin gene was determined and verified by the following experiments.Chromatin immunoprecipitation(Ch IP)experiments were utilized to further verify whether the selected TF can bind to Prestin gene.Small interfering RNA(si RNA)were peformed to selected the best specificity fragement with the effective silencing effect.Following by comparing with the expression change of Prestin between the treated group transfected with si RNA fragement and the control group to judge the regulational effect of the TF on Prestin gene.Results The expression level of Prestin protein in HEI-OC1 cells decreased with the increase of concentrations of t-BHP in the oxidative stress(24 h: F=201.541,P<0.05;48 h: F=151.169,P<0.05),while Prestin m RNA increased(24 h: F=38.709,P<0.05;48 h: F=59.125,P<0.05),and the level of Prestin protein in cells cultured for 48 h was higher than that for 24 h when the concentrations of t-BHP were the same.The reverse Ch IP assay identified 183 kinds of proteins that might bind to the Prestin gene,among them 8 TFs are characterized by the transcriptional function.In a state of oxidative stress,the expression of AP-2? m RNA showed the strongest correlation with Prestin m RNA.Ch IP-q PCR assay verified AP-2? did associate with the transcriptional start site-1441 of Prestin gene,and si RNA experiment demonstrated Prestin both at m RNA and protein level were up-regulated when AP-2? was inhibited in HEI-OC1 cells(m RNA: t=-279.292,P<0.05;protein: t=-44.548,P<0.05),it meant that TF AP-2? plays a negative regulation role in the expression of Prestin gene.The higher the concentrations of t-BHP were,the lower the expression levels of AP-2? m RNA and AP-2? protein were(P<0.05).Conclusion The research found that Prestin protein was down-regulated in HEI-OC1 cells under oxidative stress,the reason was that protein was damaged by ROS produced,Prestin m RNA presented an increase in feedback.AP-2? is an important TF modulating the expression of Prestin gene,and it can bind to the promoter region of Prestin gene,moreover,AP-2? negatively regulated its expression.The results implied that the expression of Prestin m RNA in HEI-OC1 cell was enhanced to compensate for the decrease in protein level in oxidative stress.Namely under the condition of oxidative stress Prestin expresses in a manner of compensatory mechanism,TF AP-2? is one of the important TFs that suppresses the transcription of the Prestin gene,and the inhibition effect of AP-2? on the transcription of the Prestin gene in oxidative stress state is weakened.The founding not only enriches the molecular regulation mechanism of Prestin,but also provides a new perspective for the mechanism of NIHL.Furthermore,AP-2? may also be a target for repairing the damage of the cochlea outer hair cells of NIHL.