Metformin (Sigma-Aldrich), DMEM/F-12 medium and Trizol (Invitrogen-Gibco), Nu serum™ and ITS + premix [including insulin (6.25 µg/mL), transferrin (6.25 µg/mL), selenium (6.25 ng/mL), bovine serum albumin (1.25 mg/mL) and linoleic acid (5.35 µg/mL)] (BD Biosciences), reverse transcription kit and MTS (Promega), Power SYBR® Green PCR Master Mix (ABI) USA, cortisol chemiluminescence immunoassay kit (Siemens), aldosterone radioimmunoassay kit (Beijing North Biotechnology Research Institute).
The human adrenocortical carcinoma cell line NCI-H295R cells were provided by the Cell Center of the Institute of Basic Medicine, Peking Union Medical College. NCI-H295R cells were cultured in DMEM/F-12 complete medium (containing 2.5% Nu-serum™, 1% ITS + premix, 50 units/mL penicillin and 50 units/mL streptomycin), at 37℃ in an incubator supplied with humidified air. Cells were passaged by digestion with trypsin-EDTA (0.25% trypsin and 0.02% EDTA in PBS without Ca2+ and Mg2+).
Cell Proliferation Assay
Cells were plated into 96-well culture plates (6 × 104 cells/mL, 200 µL per well) in complete medium. At logarithmic growth phase, cells were incubated in fresh serum-free DMEM/F12. After serum starvation for 6 hours, cells were treated as follows: 1) Cells were treated with different concentrations of metformin (0.01 mmol/L, 0.1 mmol/L, 1 mmol/L, 5 mmol/L, 10 mmol/L, 20 mmol/L, 50 mmol/L) for 3 days. 2) Cells were treated with 20 mmol/L metformin for 12 hours, 1 day, 2 days, 3 days and 5 days respectively. Culture medium was removed and 100 µL DMEM/F12 medium and 20 µL MTS were added to each well. Cells were incubated at 37 °C for 40 min. Finally, the plates were read on a microplate reader (Bio-TEK Instruments, Vermont, USA) at 490 nm with a reference filter at 630 nm. The experiment was repeated three times.
Detection of Hormone Concentration
Cells were cultured in 24-well culture plates (4 × 105 cells/mL, 1 mL per well) in complete medium. Then cells were treated as aforementioned method in cell proliferation experiments. The culture medium was collected for measurement of cortisol (chemiluminescence assay) and aldosterone (radioimmunoassay). The experiment was repeated three times.
Real-Time Quantitative PCR
Cells were cultured in 6-well culture plates (5 × 105 cells/mL, 2 mL per well) in complete medium. At logarithmic growth phase, cells were incubated in fresh serum-free DMEM/F12 for starvation for 6 hours, and then treated with metformin (20 mmol/L) for 24 hours. Cells were lysed with Trizol reagent, and total RNA isolation and cDNA synthesis were conducted by following the standard protocol provided by the manufacturer. PCR amplification for CYP11B1 and CYP11B2 genes was conducted by using the Power SYBR ® Green PCR Master on an ABI ViiA7 PCR system. The relative gene expression levels were calculated by using the cycle threshold value and were normalized against the expression of β-actin gene. All real-time PCR was performed in triplicate for each sample. The experiment was repeated three times.
The primers used in the experiment were as follows: CYP11B1 primer: forwards 5'-AATGCGGAACTGTCGCCAGATG-3', backwards 5'-TCAGCAAGGGAAACACCGTC-3'; CYP11B2 primer: forwards 5'-ACTCGCTGGGTCGCAATG-3', backwards 5'-AGTGTCTCCACCAGGAAGTGC-3'; β-actin primer: forward-s 5'-TCCCTGGAGAAGAGCTACG-3', backwards: 5'-GTAGTTTCGTGGATGCCACA-3'.
The statistical analyses were performed by using the SPSS version 13.0 software package. Data were presented as means ± standard deviation, and significance of differences was assessed by using independent samples t-test. 𝑃<0.05 was considered to be statistically significant.