a Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital , Fuzhou , China.
b Key Laboratory of Ministry of Education for Gastrointestinal Cancer , Research Center for Molecular Medicine, Fujian Medical University , Fuzhou , China.
J Enzyme Inhib Med Chem. 2019 Dec;34(1):799-807. doi: 10.1080/14756366.2019.1587416.
mPGES-1 is a terminal rate-limiting enzyme responsible for inflammation-induced PGE2 production. The inhibition of mPGES-1 has been considered as a safe and effective target for the treatment of inflammation and cancer. However, a specific, efficient, and simple method for high-throughput screening of mPGES-1 inhibitors is still lacking. In this study, we developed a fluorescence imaging strategy to monitor the expression of mPGES-1 via CRISPR/Cas9 knock-in system. Immunofluorescence colocalisation, Sanger sequencing, RNAi, and IL-1β treatment all confirmed the successful construction of mPGES-1 reporter cells. The fluorescence signal intensity of the reporter cells treated with four conventional mPGES-1 inhibitors was considerably attenuated via flow cytometry and fluorescent microplate reader, demonstrating that the reporter cells can be used as an efficient and convenient means for screening and optimising mPGES-1 inhibitors. Moreover, it provides a new technical support for the development of targeted small molecule compounds for anti-inflammatory and tumour therapy.
mPGES-1 是一种末端限速酶，负责炎症诱导的 PGE2 产生。抑制 mPGES-1 已被认为是治疗炎症和癌症的一种安全有效的靶点。然而，仍然缺乏用于高通量筛选 mPGES-1 抑制剂的特异性、高效和简单的方法。在这项研究中，我们通过 CRISPR/Cas9 敲入系统开发了一种荧光成像策略来监测 mPGES-1 的表达。免疫荧光共定位、Sanger 测序、RNAi 和 IL-1β 处理均证实了 mPGES-1 报告细胞的成功构建。通过流式细胞术和荧光微孔板读数，报告细胞经四种常规 mPGES-1 抑制剂处理后荧光信号强度明显减弱，表明报告细胞可作为筛选和优化 mPGES-1 抑制剂的有效且便捷的手段。此外，它为开发针对抗炎和肿瘤治疗的靶向小分子化合物提供了新的技术支持。
