P450 glotm assay

CYP1A2 enzyme activity in HepG2 cells and Huh7 cells treated with lansoprazole, with or without hsa-miR-132-5p synthetic miRNA mimic, was measured using the substrate-specific P450-Glo^TM Assay (Promega, Madison, WI). To verify the specific inhibitory effects of the hsa-miR-132-5p mimic, CYP2B6 and CYP3A4 enzyme activities were also measured as negative controls. HepG2 cells and Huh7 cells were seeded at a density of 2 × 10⁴ per well in 96-well plates. After being incubated overnight, synthetic hsa-miR-132-5p mimic together with negative control miRNA mimic were transfected into HepG2 cells and Huh7 cells using Lipofectamine 2000 reagent. After incubation at 37 °C in a CO₂ incubator for 6 h, the transfection medium was exchanged with fresh medium containing lansoprazole (50 μM) and then incubated for a period of 12 h. The cells were washed with PBS twice and incubated with 50 μL of fresh culture medium containing the corresponding CYP1A2 substrate (Luciferin-1A2), CYP2B6 substrate (Luciferin-2B6), or CYP3A4 substrate (Luciferin-IPA) at 37 °C for 1 h. Then 25 μl of culture medium was transferred to a white 96-well plate together with 25 μL of luciferin detection reagent added in each well. The luminescence was measured with a BioTek Cytation 5 (BioTek, Winooski, VT) cell imaging multimode reader.