DNA甲基转移酶Dnmt1/3a与转录因子Sp1竞争性结合BACE1启动子对RPE细胞中Aβ积聚的转录调控机制研究

Amyloid beta (Aβ) is an important toxic composition beneath the retinal pigment epithelium (RPE) layer, leading to RPE degeneration and apoptosis. The results of clinical trials on drugs targeting Aβ showed efficacy only in early stage patients with Alzheimer's Disease, along with evident side effects. Thus, anti-Aβ at an early stage might be a better interventional strategy for RPE protection. Our former studies showed that the productive rate of Aβ was determined by BACE1 but the underlying mechanism required further confirmation. Meanwhile, BACE1 transcriptional levels were simultaneously regulated by Dnmt1/3a and Sp1. Based on these observations, we speculated that the enhanced transcription of BACE1, regulated by Dnmt inactivation and increased Sp1 binding, contributed to the overexpression and accumulation of Aβ. To confirm this hypothesis, we established the photo-oxidative model of RPE cells and eventually Aβ levels were quantified after BACE1 inhibition or activation by gene transfection. The levels of differential methylation of CpG islands within the BACE1 promoter after photo-oxidation were quantified by mass spectrometry. The overlapping combinding sites of Dnmt and Sp1 within the BACE1 promoter were predicted by bioinformatics analyses. Chromatin immunoprecipitation assay was used to verify the changes in the Sp1 combinational levels binding to BACE1 promoter. In this way, we aimed to clarify the positive/negative regulation of Dnmt and Sp1 on BACE1 transcription. Finally, the effect of Sp1 inhibitor was investigated to explore its role as new anti-Aβ target drug in retina.

β淀粉样肽 (Aβ)是干性年龄相关性黄斑变性(AMD)中视网膜色素上皮细胞(RPE)下重要的毒性积聚物，导致其变性凋亡。直接靶向Aβ的艾滋海默药物临床试验结果显示其仅对早期患者有效且存在副作用。故从始动环节干预Aβ是较好的RPE保护策略。前期研究发现Aβ的产生受BACE1调控，但该机制尚待讨论。同时，BACE1的转录受甲基转移酶Dnmt1/3a及Sp1调控。故推测，BACE1转录活性增强可导致Aβ生成、沉积增加，该异常过程可能由甲基转移酶失活协同Sp1结合增加共同调控。为证实该假说，拟在RPE光氧化应激模型中，采用基因转染技术调节BACE1水平，检测Aβ含量改变；质谱法检测BACE1启动子甲基化水平；生物信息学预测共同结合位点；染色质免疫沉淀技术检测Sp1与BACE1结合程度，明确甲基转移酶及Sp1正/逆向调控机制；最后体内外阻断Sp1，检测BACE1及Aβ含量，为新药研发寻找有效靶点。

近年大量研究发现β淀粉样蛋白（Aβ）在视网膜病变中起到重要作用，揭示了年龄相关黄斑变性（AMD）的潜在致病机制。然而，AMD患者Aβ沉积的机制未明。β-淀粉样蛋白前体蛋白裂解酶1（BACE1）是Aβ产生的限速酶，在脑内Aβ沉积中起重要作用。在当前的研究中，我们旨在阐明BACE1的调控机制，并使用脂褐素荧光团A2E介导的光氧化模型探索潜在的药物靶点。在该模型中，Aβ1-40和Aβ1-42水平与BACE1表达增强呈正相关。这些变化与BACE1基因启动子内特定基因座的低甲基化和DNA甲基转移酶1（Dnmt1）的水平降低有关。此外，我们注意到BACE1启动子内差异甲基化CpG岛和转录因子Sp1结合位点具有重叠区域。我们采用染色质免疫沉淀（ChIP）分析来验证DNMT1减少的BACE1启动子甲基化使Sp1和BACE1启动子之间的结合增加，从而进一步增强BACE1转录。Mithramycin A（MTM）对Sp1的抑制作用可能下调BACE1的表达，并减轻RPE屏障的形态和功能损害。我们的结果首次显示了光氧化后转录因子Sp1和DNMT1对BACE1的竞争调节，并证实了MTM对RPE细胞的潜在新保护作用。项目研究成果在Neurochemistry International等期刊发表论文2篇。

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