HEC-1α cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and Ishikawa cells were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). HEC-1α cells were cultured in a 5% CO₂ incubator at 37°C using McCoy's 5a medium (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and Ishikawa cells were cultured in a 5% CO₂ incubator at 37°C using Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum.

A total of 53 endometrial carcinoma tissues and eight normal endometrial tissues were obtained during surgery or endometrial curettage at Soonchunhyang University Cheonan Hospital, (Cheonan, Korea) between January 1998 and December 2006. All endometrial cancers exhibited an endometrioid adenocarcinoma histology. Tumor stage was assessed according to the International Federation of Gynecology and Obstetrics staging system and differentiation grade was assessed according to the World Health Organization classification 1–3 system. The clinicopathological characteristics of the patients are described in Table I. Written informed consent was obtained from all patients, and the study was approved by the Institutional Review Board of Soonchunhyang University Cheonan Hospital.

RT-PCR was performed in order to detect the expression of RUNX3 mRNA in the endometrial cancer cell lines, two normal endometrium and cancer tissues. Total RNA was extracted using a QIAamp RNA kit (Qiagen, Inc., Valencia, CA, USA). Reverse transcription was performed using 5 µg total RNA with the Superscript II First-Strand Synthesis system (Thermo Fisher Scientific, Inc.) using oligo(dT) primers. The PCR components were as follows: 0.5 µl forward primer (10 pmol), 0.5 µl reverse primer (10 pmol), 1 µl cDNA, 2 µl 2.5 mM dNTPs, 3 µl of 5XQ buffer, 1 U Taq DNA polymerase (Qiagen, Inc.) and dH₂O to reach a final volume of 30 µl. The primer pair for the human RUNX3 forward, 5′-AGACAGCCTGGGCTGGTAAA-3′ and reverse, 5′-TCAGATGAGTGCAGCAGGTG-3′; and GAPDH forward, 5′-CTTAGCACCCCTGGCCAAG-3′ and reverse, 5′-GATGTTCTGGAGAGCCCCG-3′. The PCR conditions were as follows: Initial denaturation for 15 min at 95°C; 35 cycles of denaturation at 95°C for 30 sec, annealing at 63°C for 30 sec and extension at 72°C for 30 sec; and a final extension for 5 min at 72°C. PCR was carried out using a thermal cycler (PTC-200; MJ Research, Inc., Waltham, MA, USA). PCR products were evaluated by electrophoresis on ethidium bromide-stained 2% agarose gels and by digital capillary electrophoresis.

The Endometrial cancer tissue were embedded in paraffin following fixation in 10% neutral buffered formalin overnight at room temperature and sectioned at 4 µm. Immunohistochemical staining was performed to identify protein expression within the endometrial cancer tissues. To facilitate antigen retrieval, the sections were pretreated using heat-mediated antigen retrieval with sodium citrate buffer (0.01 M, pH 6.0) by boiling in a microwave for 20 min, cooling at room temperature for at least 1 h, and rinsing with distilled water three times for 3 min each. To block endogenous peroxidase, the sections were incubated in 3% hydrogen peroxide in methanol at room temperature for 20 min. Next, sections were washed three times with distilled water, soaked in PBS pH 7.4 for 3 min, and then incubated with rabbit polyclonal antibody against human RUNX3 (39301; 1:100; Active Motif, Inc., Carlsbad, CA, USA) at room temperature for 1 h. Sections were washed with PBS three times for 3 min each and then were incubated with secondary antibody EnVision HRP-Labelled Polymer anti-rabbit (K4002; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 30 min at room temperature. Thereafter, the sections were washed three times with PBS and reacted with the chromogen diaminobenzidine, followed by washing with distilled water. Sections were then counterstained with hematoxylin and mounted with DPX mounting medium. The slides were evaluated by a trained pathologist using an optical light microscope. Depending on the % of stained cells, quantification was performed according to the following classification: Staining intensity 0, no stained cancer cells; +1, positive in <33% of cancer cells; +2, positive in 33–66% of cancer cells; and +3, positive in >66% of cancer cells.

A G-spin Genomic DNA Extraction kit (Intron Biotechnology, Inc., Seongnam, Korea) was used for DNA extraction from cell lines and tumor tissues. To obtain DNA from paraffin blocks containing tumor tissues, microdissection was conducted as follows. First, 5–7 µm sections were cut from the paraffin blocks. Following deparaffinization, the blocks were hydrated through a graded series of alcohols to distilled water. Next, the sections were dried at room temperature. The tumor tissues were then scraped with a 24-gauge needle from dried tissue under a microscope at magnification, ×40. The collected tissue was placed in 50 µl of lysis buffer (500 mM KCl, 150 mM Tris-HCl pH 8.0, 15 mM MgCl₂ and 1.5% Tween-20) containing proteinase K (0.5 mg/l). Samples were mixed well by vortexing and incubated for 24 h at 55°C. Subsequently, DNA was extracted using the G-spin Genomic DNA Extraction kit and bisulfite modification, which converts unmethylated CpG cytosine to uracil, was carried out using an Epitech Bisulfite kit (Qiagen, Inc.).

In order to determine the methylation status of the RUNX3 promoter, MS-PCR was performed. Primers specific for methylated DNA (M; forward, 5′-TATTCGTTAGGGTTCGTTCGT-3′ and reverse, 5′-AAACAACCACGAAAAACGAC-3′) and unmethylated DNA (U; forward, 5′-AAGTGGGAAAGTAGAAGTGGTG-3′ and reverse, 5′-CCAAACAAACTACAAACAACCA-3′) were used. The PCR conditions were as follows: Initial denaturation for 15 min at 95°C; 35 cycles of denaturation at 95°C for 15 sec, annealing at 58°C for 50 sec and extension at 72°C for 60 sec, and a final extension for 5 min at 72°C. PCR was carried out in a thermal cycler (PTC-200; MJ Research, Inc.). PCR products were then subjected to capillary electrophoresis or electrophoresis on ethidium bromide-stained 2% agarose gels. The results were verified using a digital image analysis system. The positive control DNA template was a universally methylated DNA (S7821), and the negative control DNA template was a universally unmethylated DNA (S7822; both EMD Millipore, Billerica, MA, USA).

Because methylation is a reversible modification, the DNA methyltransferase inhibitor ADC (Sigma-Aldrich; Merck KGaA) was used to examine whether gene expression was inhibited by promoter methylation. HEC1-α cells were treated with ADC at different concentrations (0, 0.5, 1 or 5 µM) for 3 days. Cells were then harvested for analysis of RUNX3 mRNA expression.

To evaluate the biological function of RUNX3, the viability of HEC1α cells was analyzed following treatment with ADC using an MTT assay (Sigma-Aldrich; Merck KGaA), according to the manufacturer's instructions. Cell survival was calculated as the optical density value at 545 nm ×100.

All statistical analyses were carried out with SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA) using χ², students-t test and one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

As described in Table I, the average age of the patients was 59.3±11.8 years, and the histological grades were grade 1 or 2 in 39 patients (73.6%) and grade 3 in 14 patients (26.4%). A total of 48 patients underwent staging surgery, including hysterectomy, bilateral salpingo oophorectomy, pelvic lymphadenectomy, multiple peritoneal biopsy, and peritoneal washing cytology. According to the surgical pathology reports, clinicopathological factors were as follows: Stage I in 35 patients (72.9%); stage III in 13 patients (27.1%); myometrial invasion, less than half in 21 patients (43.7%); and myometrial invasion, more than half in 27 patients (56.3%).

RUNX3 mRNA was expressed in Ishikawa cells but not in HEC1-α cells (Fig. 1A). In addition, results from MS-PCR revealed that the RUNX3 promoter region was methylated in HEC1-α cells but not in Ishikawa cells (Fig. 1B). RUNX3 mRNA expression was absent in two cases of fresh endometrial cancer tissue but was present in two cases of fresh normal endometrial tissues (Fig. 1A).

Immunohistochemical staining of tumor sections from 53 cases of endometrioid adenocarcinoma revealed that RUNX3 protein was expressed in 26 cases (49.1%). Normal endometrial tissues surrounding the endometrial cancer exhibited strong RUNX3 protein expression (+3) and were selected as the positive control group (data not shown). RUNX3 protein expression was observed in the cytoplasm and was widely spread evenly throughout the samples (Fig. 2). Among 39 cases with grade 1~2 tumors, 16 cases (41.0%) demonstrated negative staining, 20 cases (51.3%) demonstrated +1 expression and only 3 cases (7.7%) demonstrated +2 expression, among 14 cases with grade 3 tumors, 11 cases (78.5%) demonstrated negative staining, 2 cases (14.2%) demonstrated +1 expression and only 1 case (7.1%) demonstrated +2 expression (Table I). Of the 53 patients, 48 underwent surgery, including total hysterectomy, bilateral salpingo oophorectomy, multiple peritoneal biopsy, pelvic lymphadenectomy, and peritoneal washing cytology. Therefore, more detailed staging information, as well as myometrial and lymph node invasion status, was available for these samples (Table I). Among 35 cases with stage I tumors, 11 cases (31.4%) demonstrated negative staining, 20 cases (57.1%) demonstrated +1 expression and 4 cases (11.4%) demonstrated +2 expression, among 13 cases with stage III tumors, only 1 case (7.6%) demonstrated +1 expression (Table I).

The RUNX3 expression pattern was compared to age, stage, grade, myometrial invasion and lymph node metastasis of the patients. The statistical analysis revealed that the higher histological grades were significantly associated with lower RUNX3 protein expression (P<0.05; Table I) and that loss of RUNX3 protein expression was correlated with advanced stage (P<0.05; Table I).

Methylation of the RUNX3 promoter region was observed in 33 of 53 cases (62.3%) of endometrial cancer and in one of eight cases (12.5%) of normal endometrial tissues (Fig. 3). Methylation was more frequently observed in tumor tissues than in normal tissues (P<0.05). However, there was no significant correlation between RUNX3 promoter methylation and clinicopathological prognostic factors (Table II).

RUNX3 mRNA expression was increased following 72 h of treatment with increasing concentrations of ADC in HEC1-α cells (Fig. 4), which were demonstrated in the present study to be negative for RUNX3 protein expression and positive for methylation of the RUNX3 promoter (Fig. 1). This result confirmed that silencing of RUNX3 in HEC1-α cells occurred by methylation of the RUNX3 promoter region.

Cell viability was evaluated by MTT assay in HEC1-α cells following treatment with ADC (0, 0.5, 1, 2.5 and 5 µM) for 72 h. The results demonstrated that ADC treatment inhibited cell growth in a concentration and time-dependent manner (Fig. 5).