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Establishment of a specific in vivo Cu(Ⅰ) reporting system based on metallothionein screening.
Metallomics ( IF 2.9 ) Pub Date : 2021-07-02 , DOI: 10.1093/mtomcs/mfab035
Mingjun Ma 1 , Botao Qiu 1 , Jing Jin 1 , Jiaru Wang 1 , Yuzhe Nie 1 , Yang Liang 1 , Ze Yu 1 , Chun-Bo Teng 1
Affiliation  

Copper is one of the indispensable trace metal elements in organisms, but excess copper means cytotoxicity. Cells protect themselves by storing excess copper in copper-binding proteins. Metallothioneins (MTs) are a group of low-molecular-weight, cysteine-rich proteins, which are well known for sensing and binding the overcharged Zn(Ⅱ), Cd(Ⅱ), and Cu(Ⅰ) in cells. However, there are only few reports on MTs that can specifically respond to intracellular copper ions in mammals in real-time. Here, we screened copper-response MTs in pancreatic cancer cells through data-mining, RNA-seq, and qPCR analysis. We found that MT1E, MT1F, and MT1X mRNA were significantly upregulated after exogenous copper ion induction. By constructing the stable cell lines with MT1E, MT1F, or MT1X promoter-driven EGFP as reporters, we found that only PMT1F-EGFP could specifically and stably report the intracellular Cu(Ⅰ) changes in multiple cell lines including Panc-1, 8988T, 293T, HepG2, and normal hepatic cells, indicating that PMT1F-EGFP is an ideal in vivo Cu(Ⅰ) reporter. Using the PMT1F-EGFP reporter, we found that MEK inhibitors (U0126) and Astragaloside IV could significantly increase intracellular copper ions. According to these results, PMT1F-EGFP reporter can sense intracellular copper change and can be used to screen copper-target drugs and study copper-related cellular physiology and pathology.

中文翻译:

基于金属硫蛋白筛选的特异性体内Cu(Ⅰ)报告系统的建立

铜是生物体内不可缺少的微量金属元素之一,但过量的铜意味着细胞毒性。细胞通过将多余的铜储存在铜结合蛋白中来保护自己。金属硫蛋白 (MTs) 是一组低分子量、富含半胱氨酸的蛋白质,以感知和结合细胞中过度充电的 Zn(Ⅱ)、Cd(Ⅱ) 和 Cu(Ⅰ) 而闻名。然而,关于 MT 可以实时特异性响应哺乳动物细胞内铜离子的报道很少。在这里,我们通过数据挖掘、RNA-seq 和 qPCR 分析筛选了胰腺癌细胞中的铜反应 MT。我们发现外源性铜离子诱导后 MT1E、MT1F 和 MT1X mRNA 显着上调。通过构建以 MT1E、MT1F 或 MT1X 启动子驱动的 EGFP 作为报告基因的稳定细胞系,我们发现只有PMT1F-EGFP能够特异性、稳定地报告包括Panc-1、8988T、293T、HepG2和正常肝细胞在内的多个细胞系的细胞内Cu(Ⅰ)变化,表明PMT1F-EGFP是一种理想的体内Cu (一)记者。使用 PMT1F-EGFP 报告基因,我们发现 MEK 抑制剂 (U0126) 和黄芪甲苷可以显着增加细胞内铜离子。根据这些结果,PMT1F-EGFP报告基因可以感知细胞内铜的变化,可用于筛选铜靶向药物和研究铜相关的细胞生理和病理学。我们发现MEK抑制剂(U0126)和黄芪甲苷可以显着增加细胞内铜离子。根据这些结果,PMT1F-EGFP报告基因可以感知细胞内铜的变化,可用于筛选铜靶向药物和研究铜相关的细胞生理和病理学。我们发现MEK抑制剂(U0126)和黄芪甲苷可以显着增加细胞内铜离子。根据这些结果,PMT1F-EGFP报告基因可以感知细胞内铜的变化,可用于筛选铜靶向药物和研究铜相关的细胞生理和病理学。
更新日期:2021-07-02
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