Purpose: To determine the mechanism of EPE in downregulating TYMS in MPM cancer. Methods: The TYMS mRNA expression with epithelial-to-mesenchymal transition biomarkers and nuclear factor SP1 was assessed using the GEO database in a data set of MPM patients (GSE51024). Invasive MPM cell lines were in vitro models for the investigation of TYMS expression after EPE treatment. Thetymspromoter SP1 binding sequences were determined using Genomatix v 3.4 software Electrophoretic mobility shift and dual-luciferase reporter assays revealed specific SP1 motifs in the interaction of EPE and reference compounds. Chromatin immunoprecipitation and Re-ChIP were used for the co-occupancy study. Results: In MPM patients, a positive correlation of overexpressed TYMS with mesenchymal TWIST1, FN1 and N-cadherin was observed. EPE and its major components, gallic and ellagic acid (GA and EA, respectively), downregulated TYMS in invasive MPM cells by interacting with particular SP1 motifs on thetymspromoter. The luciferase constructs confirmed the occupation of two SP1 regulatory regions critical for the promotion of TYMS expression. Both EPE and reference standards influenced SP1 translocation into the nucleus. Conclusion: EPE components reduced TYMS expression by occupation of SP1 motifs on thetymspromoter and reversed the EMT phenotype of invasive MPM cells. Further in-depth analysis of the molecular docking of polyphenol compounds with SP1 regulatory motifs is required.
目的：探究EPE下调MPM癌细胞中TYMS表达的机制。方法：利用GEO数据库中MPM患者数据集（GSE51024），评估TYMS mRNA表达与上皮-间质转化生物标志物及核因子SP1的关联。采用侵袭性MPM细胞系作为体外模型，研究EPE处理后TYMS的表达变化。通过Genomatix v3.4软件分析tyms启动子SP1结合序列，并采用电泳迁移率变动实验和双荧光素酶报告基因检测验证EPE及其对照化合物与SP1基序的特异性相互作用。染色质免疫共沉淀及再染色质免疫共沉淀技术用于共占据位点研究。结果：在MPM患者中观察到过表达的TYMS与间质标志物TWIST1、FN1及N-钙黏蛋白呈正相关。EPE及其主要成分没食子酸与鞣花酸通过作用于tyms启动子上的特定SP1基序，下调侵袭性MPM细胞中TYMS表达。荧光素酶报告基因构建体证实了两个对TYMS表达至关重要的SP1调控区域被占据。EPE与对照标准物均能影响SP1向细胞核的转位。结论：EPE活性成分通过占据tyms启动子上的SP1基序降低TYMS表达，并逆转侵袭性MPM细胞的上皮-间质转化表型。未来需对多酚类化合物与SP1调控基序的分子对接机制进行深入分析。
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