Cell culture
The human HCC cell lines Hep3B (ATCC, Manassas, VA, USA; HB-8064) and HepG2 (ATCC, HB-8065) and human colon cancer cell line HT-29 (ATCC, HTB-38) were cultured on 100-mm culture plates (Falcon, Corning, NY, USA) in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, Irvine, UK) containing 10% fetal calf serum (FCS; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and penicillin (100 μg/mL; GE Healthcare Life Sciences, Pasching, Austria) at 37°C in a humidified chamber with 5% CO2. 5-Aza (Sigma) was dissolved in deionized water (1 mM) and diluted to a concentration of 0, 5, or 10 μM in the culture fluid; cells were treated with different concentrations of 5-aza for 24, 48, or 72 h.
Plasmid construct
The plasmids for the transient expression assay to examine basal promoter activity of the human UCP2 gene were constructed using the SEAP reporter system (TaKaRa, Tokyo, Japan) following the manufacturer’s protocol. The following primers with appropriate restriction sites were used to amplify the promoter regions [23]: sense primer sequence, 5′-GGTACCTCAAGATAACTGGTATGCCTTGT-3′, and antisense primer sequence, 5′-GAATTCTCATACTATGTGTCCGAGCCGCA-3′. PCR conditions were 40 cycles of 30 s at 94°C, 30 s at 60°C, and 3 min at 72°C, with a final extension of 10 min at 72°C. The PCR product size of UCP2 promoter was 2,960 bp. The PCR product was ligated into the KpnI/EcoRI site of the polylinker region of the SEAP2 basic vector.
Analysis of basal promoter activity of the human UCP2 gene
To examine basal promoter activity of the human UCP2 gene, transient expression assay of the UCP2 promoter SEAP construct was performed in Hep3B, HT-29, and HepG2 cell lines. The cells were cultured at a density of 1 × 105 cells in 35-mm dishes and DMEM containing 10% FCS. After seeding, the dishes were washed extensively to remove non-adherent cells and the medium was replaced. On the second day, transfection was performed with FuGENE 6 transfection reagent (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s protocol. The plasmid (1 μg) consisting of the human UCP2 promoter region was fused to the SEAP basic vector and 1 μg of the SEAP control vector. After transfection, the medium was replaced and the cells cultured for an additional day. The supernatant was collected from each sample culture and SEAP activity measured.
Total RNA extraction and cDNA synthesis
Cells were homogenized with 1 mL of TRIzol Reagent (Ambion, Carlsbad, NM, USA), mixed with 0.3 mL of chloroform, and centrifuged at 12,000 rpm for 15 min at 4°C. The aqueous layer was transferred to a new tube and 0.6 mL of isopropanol added. The tubes were inverted several times and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was removed and the RNA pellet washed with 70% alcohol. The RNA pellet was briefly air-dried and dissolved in diethyl dicarbonate-treated water. The total RNA concentration was measured using a NanoDrop (Molecular Devices, LLC, Sunnyvale, CA, USA). Complementary DNA (cDNA) was synthesized using the PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa).
PCR
PCR was performed using premix Taq (TaKaRa) and specific primers. Primer sequences used to amplify UCP2 were designed based on GenBank sequences: sense primer sequence, 5′-GCCCGGGCTGGTGGTGGTC-3′ and antisense primer sequence, 5′-CCCCGAAGGCAGAAGTGAAGTGG-3′. PCR UCP2 amplification consisted of denaturation at 94°C for 2 min, followed by 25 cycles of 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C, with a final extension for 7 min at 72°C. The PCR products were analyzed using 2% agarose gel electrophoresis; the UCP2 PCR product size was 290 bp. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the control housekeeping gene. GAPDH amplification consisted of denaturation at 94°C for 30 s, followed by 30 cycles of 30 s at 94°C, 30 s at 50°C, and 30 s at 72°C, with a final extension for 10 min at 72°C. The GAPDH sense primer sequence was 5′-ACCACAGTCCATGCCATCAC-3′ and the antisense primer sequence was 5′-TCCACCACCCTGCTGTA-3′. The PCR products were analyzed using 2% agarose gel electrophoresis; the GAPDH PCR product size was 450 bp.
Protein extraction and Western blot analysis
The 5-aza-treated cells were homogenized using a sonicator with lysis buffer containing protease inhibitors. Lysates were centrifuged at 12,000 rpm for 20 min at 4°C. Then, the protein lysates were transferred to a new tube. Total protein concentration was assessed using the BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Protein samples were boiled at 95°C for 5 min after adding 5× sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis loading buffer (25 mM Tris-HCL pH 6.8, 10% SDS, 50% glycerol, 0.5 M dithiothreitol, 0.5% bromophenol blue). After electrophoresis, the proteins were transferred to a nitrocellulose membrane and blocked with 3% skim milk (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 2 h. Membranes were washed with 1× TBS-T and incubated overnight at 4°C with specific antibodies against UCP2 (1:1,000; Integrated DNA Technologies, Science Park II, Singapore), DNMT1 (1:1,000; Integrated DNA Technologies), DNMT3a (1:1,000; Integrated DNA Technologies), DNMT3b (1:1,000; Integrated DNA Technologies), and β-actin (1:5,000; Integrated DNA Technologies). The membranes were washed with 1× TBS-T and incubated for 2 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG polyclonal antibody (Bethyl, Montgomery, AL, USA) or HRP-conjugated anti-mouse IgG polyclonal antibody (Bethyl Laboratories, Montgomery, TX, USA) at room temperature. Bands were detected using West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).
Measurement of DNMT activity
Nuclear proteins were isolated using the EpiQuik™ Nuclear Extraction Kit I (Epigentek, Brooklyn, NY, USA) from 5-aza-treated cells. After measuring the protein concentration with the BCA Protein Assay Kit (Thermo Fisher Scientific), total DNMT activity was analyzed using EpiQuik™ DNA Methyltransferase Activity/Inhibition Assay (Epigentek).
gDNA purification and bisulfite modification
Genomic DNA (gDNA) was isolated from 5-aza-treated cells using the Wizard® Genomic DNA Purification Kit (Promega Corp., Madison, WI, USA) according to the manufacturer’s instructions. Extracted gDNA was modified using the EpiTect® Bisulfite Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. During the experiment, DNA concentration was measured using a NanoDrop (Molecular Devices). Sodium bisulfite-modified DNA was stored at −15 to −30°C.
MSP and direct PCR sequencing assays
Specific methylated and unmethylated primers are required for MSP analysis. The primer design for non-sulfurized processing sequences is available on the MetPrimer site (http://www.urogene.org/methprimer2/). Specific PCR conditions such as specific methylated and unmethylated primer sequences, combined temperature, and number of cycles of target genes are presented in Table 1. Sodium bisulfite-modified DNA was analyzed using the EpiScope® MSP Kit (TaKaRa) according to the manufacturer’s instructions. The PCR products were analyzed using 3% agarose gel electrophoresis. The MSP samples were sent to Macrogen Corporation (Seoul, South Korea) for direct PCR sequencing.
Statistical analysis
All experiments were repeated five to eight times. The data were expressed as the means ± standard error of the mean. All statistical analyses were performed using analysis of variance with the Statistical Analysis System (SAS) software (SAS Institute, Cary, NC, USA); each treatment was compared using the least-squares or Duncan method. A P-value < 0.05 indicated significant differences among treatments.