Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Auerbachstrasse 112, DE-70376 Stuttgart.
Chem Biodivers. 2009 Nov;6(11):2084-91. doi: 10.1002/cbdv.200900135.
Human NADPH : cytochrome P450 oxidoreductase (POR) is encoded by a single gene on chromosome 7q11.2. This flavoprotein donates electrons derived from NADPH to a variety of acceptor proteins, including squalene monooxygenase, heme oxygenase, cytochrome b(5), and many microsomal cytochromes P450 (CYPs), which are involved in oxidative drug metabolism, steroidogenesis, and other functions. Numerous aspects related to cellular POR expression have not been systematically investigated. Interestingly, POR expression is lower compared to CYPs and may thus be limiting for monooxygenase activities, but conversely, POR knock-out in mice resulted in compensatory upregulation of CYPs. POR may also influence intracellular cholesterol and lipid homeostasis. To systematically investigate such effects, we developed specific POR gene silencing in cell lines and primary human hepatocytes by RNA interference using small interfering RNAs (siRNAs). In HepG2 cells, POR mRNA could be reduced by 95% over 4 days accompanied by reduced protein content and activity. In primary human hepatocytes, POR mRNA knock-down was less effective and more variable. Analysis of CYPs indicated induction of CYP3A4 but not CYP1A2 or CYP2D6. These results demonstrate that POR can be efficiently and almost completely silenced in HepG2 cells and, at least partially, in primary human hepatocytes. This will allow systematic studies of various consequences of POR variability in human cells.
人类 NADPH：细胞色素 P450 氧化还原酶（POR）由染色体 7q11.2 上的单个基因编码。这种黄素蛋白将 NADPH 衍生的电子捐赠给各种受体蛋白，包括角鲨烯单加氧酶、血红素氧合酶、细胞色素 b(5)和许多微粒体细胞色素 P450（CYPs），它们参与氧化药物代谢、类固醇生成和其他功能。与细胞 POR 表达相关的许多方面尚未系统研究。有趣的是，与 CYPs 相比，POR 的表达水平较低，因此可能限制单加氧酶的活性，但相反，小鼠中的 POR 敲除导致 CYP 的代偿性上调。POR 还可能影响细胞内胆固醇和脂质稳态。为了系统地研究这些影响，我们通过使用小干扰 RNA（siRNA）的 RNA 干扰在细胞系和原代人肝细胞中特异性地沉默 POR 基因。在 HepG2 细胞中，POR mRNA 可在 4 天内减少 95%，同时蛋白含量和活性降低。在原代人肝细胞中，POR mRNA 的敲低效果较差且更具可变性。对 CYP 的分析表明 CYP3A4 的诱导，但 CYP1A2 或 CYP2D6 没有诱导。这些结果表明 POR 可以在 HepG2 细胞中有效且几乎完全沉默，并且至少在原代人肝细胞中部分沉默。这将允许在人细胞中系统地研究 POR 变异性的各种后果。
