RNA-Interference Approach to Study Functions of NADPH:Cytochrome P450 Oxidoreductase in Human Hepatocytes

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.