BACKGROUND The study aimed to investigate the effect of oxidative stress on Prestin expression, and explore the transcription factors (TFs) that are involved in regulating the expression of Prestin in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells upon oxidative stress. METHODS Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression level of Prestin. Reverse chromatin immunoprecipitation (reverse ChIP) assay was performed to identify proteins that could bind to the Prestin gene. Small interfering RNA (siRNA) and chromatin immunoprecipitation (ChIP) experiments were used to further verify the results. HEI-OC1 cells were incubated with four different concentrations of tert-butyl hydroperoxide (t-BHP) for 24 h or 48 h to construct the oxidative stress model. RESULTS Oxidative stress induced Prestin increase at the mRNA level but with a concomitant decrease at the protein level. TF activating enhancer binding protein-2δ (AP-2δ) screened by reverse ChIP assay was demonstrated to bind to transcriptional start site 1441 of the Prestin promoter region and negatively regulate the expression of Prestin by siRNA and ChIP experiments. Furthermore, AP-2δ was down-regulated under oxidative stress. CONCLUSIONS In conclusion, oxidative stress inhibits the expression of Prestin protein, and the transcription mechanism is triggered to compensate for the loss of Prestin protein. AP-2δ is one of the important TFs that suppresses transcription of the Prestin gene, and AP-2δ suppression further boosted Prestin mRNA activation under oxidative stress.

中文翻译：

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AP-2δ对氧化应激下HEI-OC1细胞中Prestin基因转录的影响。

背景 本研究旨在探讨氧化应激对 Prestin 表达的影响，并探讨在氧化应激下调控 House Ear Institute-Organ of Corti 1 (HEI-OC1) 细胞 Prestin 表达的转录因子 (TFs) . 方法采用定量实时聚合酶链反应（qRT-PCR）和Western blot检测Prestin的表达水平。进行了反向染色质免疫沉淀（反向 ChIP）测定以鉴定可与 Prestin 基因结合的蛋白质。小干扰 RNA (siRNA) 和染色质免疫沉淀 (ChIP) 实验用于进一步验证结果。将 HEI-OC1 细胞与四种不同浓度的叔丁基过氧化氢 (t-BHP) 孵育 24 小时或 48 小时以构建氧化应激模型。结果 氧化应激诱导 Prestin 在 mRNA 水平上增加，但同时在蛋白质水平上降低。通过反向 ChIP 测定筛选的 TF 激活增强子结合蛋白-2δ (AP-2δ) 被证明与 Prestin 启动子区域的转录起始位点 1441 结合，并通过 siRNA 和 ChIP 实验显示负调节 Prestin 的表达。此外，AP-2δ 在氧化应激下下调。结论综上所述，氧化应激抑制了Prestin蛋白的表达，并触发了转录机制来弥补Prestin蛋白的缺失。AP-2δ是抑制Prestin基因转录的重要转录因子之一，AP-2δ抑制进一步促进了氧化应激下Prestin mRNA的活化。