Promoter Methylation Down-regulates Osteoprotegerin Expression in Ovarian Carcinoma

Materials and Methods

Cell culture and treatment. The Hs823.Tc human normal ovarian cell line as well as the SW 626, OVCAR-3, ES-2, TOV-112D, and TOV-21G ovarian carcinoma cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco's modified Eagle's medium or Roswell Park Memorial Institute 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml) (all from Gibco, Life Technologies, Grand Island, NY, USA). All cell lines were treated as previously described (1, 11, 17-19). The cells were cultured at 37°C in a humidified atmosphere of 5% carbon dioxide. The demethylating agent 5-aza-2-deoxycytidine (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate-buffered saline to a concentration of 50 mg/ml as a stock solution and stored at −20°C until use. Immediately before use, stock solutions were diluted in Roswell Park Memorial Institute 1640 medium without FBS. The cells were seeded in 6-well plates at a density of 5×10⁵ cells/ml in the media and treated with 5-aza-2-deoxycytidine (final concentration, 1 μM). The 5-aza-2-deoxycytidine was replaced with freshly prepared solution every 24 h, and the cells were harvested 96 h after the initial treatment. Control cultures were treated under similar experimental conditions in the absence of 5-aza-2-deoxycytidine (phosphate-buffered saline only).

Complementary DNA synthesis. Total RNA was isolated using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's instructions. RNase-free DNase I (Thermo Fisher Scientific, Waltham, MA, USA) treatment was carried out to remove contaminating genomic DNA from the purified total RNA obtained from the cell lines. Isolated total RNA was diluted to 1 mg/ml with sterile diethylpyrocarbonate-treated water, and 2.5 ml was added to reactions containing 1× DNase I buffer and 1 U DNase I (final volume, 10 ml). After incubation at 37°C for 30 min, the reactions were stopped by incubation at 70°C for 10 min. DNase I-treated RNA was reverse-transcribed into first-strand cDNA using the ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). One microgram of DNase I-treated RNA and 250 ng of random primers were mixed in a 0.5-ml PCR tube and brought to 11 ml with sterile diethylpyrocarbonate-treated water, heated at 65°C for 5 min, and chilled quickly on ice. Other reagents were added to the 20-ml reaction volume at the following final concentrations: 1× First-Strand Buffer, 10 mM dithiothreitol, 0.5 mM each dNTP, and 200 U Superscript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Reactions were incubated at 42°C for 1 h and heated to 70°C for 10 min, and the products were stored at −20°C. The amount of cDNA was determined using NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR). The cDNA was subjected to qRT-PCR analysis using the Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). PCR was carried out in a 20-μl reaction containing 0.5 μM each primer, 1× Thunderbird SYBR qPCR Mix (Toyobo), and 2 μl of template DNA. PCR for OPG was performed on a C1000 Thermal Cycler (Bio-Rad Laboratories) using the following reaction protocol: polymerase activation at 95°C for 1 min, followed by 40 cycles at 95°C for 10 s for denaturation, 58°C for 10 s for annealing, and 72°C for 20 s for extension. Amplification patterns were analyzed and threshold cycle numbers (Ct) for each sample were determined using CFX Manager Software (Bio-Rad Laboratories). The primer sequences used for OPG were as follows: forward, 5’-TGC TGT TCC TAC AAA GTT TA-3’, and reverse, 5’-CTT GAG TGC TTT AGT GCG T-3’. The ΔΔCt method was used to calculate relative OPG expression after normalization to the expression of β-actin (20). Amplification of OPG was confirmed by melting-curve analysis, and target amplicon size (430 bp) by agarose gel electrophoresis (Thermo Fisher Scientific). Each sample was assayed in triplicate.

Enzyme-linked immunosorbent assay (ELISA). For analysis of OPG protein production, culture media were harvested and centrifuged to remove cellular debris. Media were concentrated by centrifugal filtration at 4,000 rpm for 20 min using the Amicon Ultra-10K concentrator (EMD Millipore, Burlington, MA, USA). A commercially available ELISA kit for Osteoprotegerin (Cloud-Clone Corporation, Katy, TX, USA) was used according to the protocols provided by the manufacturer to measure the concentrations of human OPG. Each sample was assayed in triplicate.

Genomic DNA extraction and bisulfite conversion. Genomic DNA was extracted from cultured cells using the NucleoSpin Tissue kit (Macherey-Nagel GmbH & Co. KG, Duren, Germany) and quantified using a DropSense96 multichannel spectrophotometer (Trinean, Gentbrugge, Belgium). Bisulfite treatment was performed using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA) on 2 μg of DNA according to the protocols provided by the manufacturer.

Pyrosequencing. DNA methylation of OPG was determined by bisulfite pyrosequencing. Primers were designed using Pyrosequencing Assay Design Software, Version 1.0.6 (Qiagen, Valencia, CA, USA). To ensure distinct pyrosequencing signals, the previously bisulfite-modified DNA (100 ng) was amplified by 45 cycles of PCR using the PyroMark PCR Kit (Qiagen) with a biotin-labeled primer according to the protocols provided by the manufacturer. The primer sequences used were as follows: forward, 5’-GGG TTT TGT AAT TTG AGG TTT TAG AA-3’, and reverse, 5’-biotin-ACT TAT ATC TCC TCC ACC CTA AA-3’. PCR products were immobilized to Streptavidin Sepharose High-Performance (GE Healthcare, Chicago, IL, USA) beads via biotin affinity. PCR products were denatured to single-stranded DNA and annealed with sequencing primers (5’-GAT AAA GGT TTG GGA TAT ATT-3’). Complexes of bead-bound single-stranded DNA and primer were applied to a PyroMark Q96 ID pyrosequencer (Qiagen) with PyroMark Gold Q96 Reagents (Qiagen). Raw results were analyzed and visualized using Pyro Q-CpG Software, Version 1.0.0 (Qiagen). Pyrosequencing was performed for three independent bisulfite reactions, and the average methylation frequency for each CpG site was calculated.

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Figure 1.

Expression of osteoprotegerin (OPG) in ovarian carcinoma and normal ovarian cell lines and effect of demethylation on OPG. (A) OPG protein expression levels measured by enzyme-linked immunosorbent assay. *p<0.05 versus Hs823.Tc cells (normal control). (B) Normalized OPG mRNA expression ratios measured by quantitative real-time reverse-transcriptase polymerase chain reaction. *p<0.05 versus Hs823.Tc cells (normal control). (C) OPG promoter methylation status analyzed by bisulfite pyrosequencing. *p<0.05 versus Hs823.Tc cells (normal control). (D) Effect of 5-aza-deoxycytidine treatment on OPG mRNA expression. *p<0.05 versus pre-treatment level. (E) Effect of 5-aza-2-deoxycytidine treatment on OPG protein expression. *p<0.05 versus pre-treatment level. Error bars represent the standard deviations.

Statistical analysis. Data are expressed as mean±standard deviation of three independent experiments, each performed in triplicate, and are presented relative to control. Wilcoxon rank sum test was used to compare the promoter methylation frequency and the expression levels of OPG mRNA and protein between the normal ovarian cell line and ovarian carcinoma cell lines. We used the Wilcoxon signed-rank test to compare the expression levels of OPG mRNA and protein before and after 5-aza-2-deoxycytidine treatment in each ovarian carcinoma cell line examined. Statistical analyses were performed using IBM SPSS Statistics for Windows, version 20 (IBM Corporation, Armonk, NY, USA). A p-value <0.05 was considered statistically significant.