Down-regulation of B-Cell Translocation Gene 1 by Promoter Methylation in Colorectal Carcinoma

Materials and Methods

Cell culture and treatment. The human normal colonic epithelial cell line CCD 841 CoTr and the human CRC cell lines HCT 116, HT-29, SW 480, and SW 620 were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated foetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml; Gibco, Life Technologies, Grand Island, NY, USA) (25). All cell lines were cultured at 37°C in a humidified atmosphere of 5% carbon dioxide. The demethylating agent 5-aza-deoxycytidine (5-aza-CdR) was obtained from Sigma-Aldrich (St. Louis, MO, USA), dissolved in phosphate-buffered saline (PBS) to a concentration of 50 mg/ml as a stock solution, and stored at −20°C until use.

Cell lines were seeded in 6-well plates at a density of 5×10⁵ cells/mL in the media and treated with 1 μM (final concentration) of 5-aza-CdR for 72 h. Freshly prepared 5-aza-CdR was replaced every 24 h and cells were harvested at 96 h after initial treatment. Control cultures were treated under similar experimental conditions in the absence of 5-aza-CdR (final concentration (PBS only).

cDNA synthesis. RNase-free DNase I treatment was carried out to remove contaminating genomic DNA from 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 at 70°C for 10 min. DNase I-treated RNA was reverse-transcribed into first-strand cDNA using random primers. DNase I-treated RNA (1 μg) and random primers (250 ng) were mixed in a 0.5-ml polymerase chain reaction (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, heated to 70°C for 10 min, and the products were stored at −20°C.

Quantitative reverse-transcriptase (qRT)-PCR. Total RNA was isolated using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) and used for cDNA synthesis with a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). The amount of cDNA was determined spectrophotometrically. The cDNA was used for qRT-PCR using the Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) with a C1000 Thermal Cycler (Bio-Rad Laboratories). PCR was carried out in a 20-μl reaction containing 0.5 μM of each primer, 10 μl of 2× SsoAdvanced SYBR Green Supermix (Bio-Rad Laboratories), and 2 μl of template DNA. PCRs for BTG1 and β-actin were initiated with a denaturing step at 95°C for 3 min, followed by 40 cycles at 95°C for 30 s and 60°C for 30 s. Amplification patterns were analysed and threshold cycle numbers (Ct) for each sample were determined using CFX Manager Software (Bio-Rad Laboratories). The ΔΔCt method was used to calculate relative target gene expression after normalization to expression of β-actin (24). The primer sequences used for BTG1 were as follows: forward: 5’-CAA GGG ATC GGG TTA CCG TTG T-3’; reverse: 5’-AGC CAT CCT CTC CAA TTC TGT AGG-3’. The ΔΔCt method was used to calculate relative target gene expression after normalization to expression of β-actin (26). Amplification of the target gene was confirmed by melting-curve analysis and target amplicon size was confirmed by agarose gel electrophoresis. Each sample was assayed in triplicate.

Western blot analysis. Whole-cell lysates were prepared in radioimmunoprecipitation assay buffer (50 mM Tris-hydrogen chloride, pH 8, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulphate) containing protease inhibitors (cOmplete Protease Inhibitor Cocktail Tablet; Roche Applied Science, Basel, Switzerland), and cleared by microcentrifugation (10,000 × g for 20 min at 4°C). The resulting lysate was assessed for protein concentration, and 20-30 μg of each protein sample was resolved using 12% sodium dodecyl sulphate-protein gel electrophoresis gel (Bio-Rad Laboratories) and electroblotted onto nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). After 1-h incubation in blocking solution (5% non-fat dry milk in Tris-buffered saline with Tween; Pierce, Rockford, IL, USA), the membranes were exposed to the following appropriate primary antibodies overnight at 4°C: anti-BTG1 (polyclonal, diluted 1:200; Abcam, Cambridge, MA, USA) and anti-α-tubulin (diluted 1:500; Abcam). The blots were washed three times in Tris-buffered saline with Tween and incubated with the horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Beverly, MA, USA) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence reagent (iNtRON Biotechnology, Seongnam, Republic of Korea).

Methylation-specific PCR. Genomic DNA was extracted from the cell lysate using a NucleoSpin Tissue kit (Macherey-Nagel, Dueren, Germany) and treated with sodium bisulphite using an EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA). During the modification, unmethylated cytosines of the genomic DNA are converted to uracils, while methylated cytosines remain unchanged. The bisulphite-modified DNA was subjected to PCR using primer pairs that specifically amplified either methylated or unmethylated sequences of BTG1. The following primers specific for methylated BTG1 were used (27): MSP1 (−149 to −289), 5’-GTT TTT AAG TTA AAA GGA AGG AAG TC-3’ (sense) and 5’-ATA TCA AAA AAT ATT AAA AAT CAC GCA-3’ (antisense); MSP2 (−517 to −645), 5’-TTT GAG GAG TTA GTT ATC GAG ATT C-3’ (sense) and 5’-AAA TAA ATA AAA ACC GCC TAA CG-3’ (antisense). The following primers specific for unmethylated BTG1 were used: USP1 (−149 to −289), 5’-GTT TTT AAG TTA AAA GGA AGG AAG TTG T-3’ (sense) and 5’-ATA TCA AAA ATA TTA AAA ATC ACA CA-3’ (antisense); USP2 (−517 to −645), 5’-TGA GGA GTT AGT TAT TGA GAT TTG G-3’ (sense) and 5’-AAA TAA ATA AAA ACC ACC TAA CAC A-3’ (antisense). Methylation-specific PCR was performed in 20-μl mixtures for 40 cycles using HotStarTaq DNA polymerase (Qiagen, Hilden, Germany). The same unconverted genomic DNAs were used in the PCR assays as negative controls.

Tissue specimens. This study was reviewed and approved by the Institutional Review Board of Myongji Hospital, Goyang, Republic of Korea (2017-11-001). CRC tissue samples were obtained from 114 consecutive patients who underwent surgery for primary adenocarcinoma of the colorectum. Twenty normal colonic mucosa samples obtained from patients without CRC were used as controls. Corresponding tissue samples from hepatic CRC metastases were also obtained from 79 patients who met the criteria for hepatic resection with curative intent (25, 28-30). In this study. no patient underwent preoperative neoadjuvant chemotherapy or neoadjuvant concurrent chemoradiation therapy.

The tissues resected by surgeons were initially examined by pathologists before fixation in 10% neutral-buffered formalin. After fixation for 12-24 h, the tissues were thoroughly examined macroscopically and sectioned. After processing with an automatic tissue processor, the sections were embedded in paraffin blocks. Four-micrometre-thick slices were cut from each formalin-fixed, paraffin-embedded (FFPE) tissue block using a rotary microtome and stained with hematoxylin and eosin using an automatic staining instrument. After staining, the slides were covered with a glass coverslip and sent to two Board-certified pathologists. The pathologists independently examined all available hematoxylin and eosin-stained slides by light microscopy, made definitive pathological diagnoses, and selected the most representative slide from each case for immunohistochemical staining.

Immunohistochemical staining. BTG1 protein expression was assessed by immunohistochemistry using the Bond Polymer Intense Detection System (Vision BioSystems, Mount Waverley, Victoria, Australia) following the manufacturer's instructions (25, 28, 31-38). In brief, the 4-μm sections of FFPE tissue were deparaffinized with Bond Dewax Solution (Vision BioSystems) and an antigen retrieval procedure was performed using Bond Epitope Retrieval Solution (Vision BioSystems) for 30 min at 100°C. Endogenous peroxidases were quenched with hydrogen peroxide for 5 min. Sections were incubated for 15 min at ambient temperature with rabbit polyclonal antibody to BTG1 (diluted 1:100; Abcam). A biotin-free polymeric horseradish peroxidase-linker antibody conjugate system was used with a Bond-maX automatic slide stainer (Vision BioSystems), and visualization was performed using 1 mM 3,3’-diaminobenzidine, 50 mM Tris-hydrogen chloride buffer (pH 7.6), and 0.006% hydrogen peroxide. Sections were counterstained with hematoxylin. To minimize inter-assay variation, positive and negative control samples were included in each run. The positive control was normal colonic tissue. The negative control was prepared by substituting non-immune serum for the primary antibody; no detectable staining was evident.

Interpretation of immunohistochemical staining. Immunostaining was analysed by two independent pathologists. The BTG1 staining intensity was graded as: 0: absent, 1: weak, 2: intermediate, or 3: strong. The staining proportion was graded as: 0: 0%, 1: 1-49%, 2: 50-74%, or 3: 75-100%. The subcellular location of BTG1-positive signals (nuclear or cytoplasmic) was also estimated. The final score was calculated as the product of the staining intensity and proportion scores, resulting in final scores of 0 (negative), 1-2 (weak), and 3-9 (positive) (25, 27, 34, 35, 39, 40). Disagreements between the two pathologists were resolved by consensus.

Statistical analysis. We used the Wilcoxon test to compare the expression levels of BTG1 mRNA before and after 5-aza-CdR treatment. Chi-squared or Fisher's exact tests were performed to compare BTG1 immunoreactivity among the normal colonic mucosa, primary CRC, and metastatic CRC tissue samples. Statistical analyses were performed using PASW Statistics for Windows (version 18.0; Armonk, NY, USA). p-Values less than 0.05 were considered statistically significant.