The human ovarian cancer cell line SK-OV-3 was purchased from the American Type Culture Collection (ATCC; no. HTB-77; Manassas, VA, USA) and A2780 was purchased from the European Collection of Cell Cultures (cat no. 93112519; Wiltshire, UK).

SK-OV-3 cells were cultured in McCoy's 5A medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Gibco-BRL, Rockville, MD, USA) in a 95% humidified air and 5% CO₂ atmosphere at 37°C.

A2780 cells were cultured in RPMI-1640 medium (Welgene, Inc., Gyeongsangbuk-do, Korea) containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Gibco-BRL) in a 95% humidified air and 5% CO₂ atmosphere at 37°C.

All procedures for handling and euthanizing the animals used in this study were performed in strict compliance with the guidelines of the Korean Animal Protection Law and were approved by the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University School of Medicine. SK-OV-3 cells (2×10⁶) suspended in culture media were intraperitoneally injected into 10 female nude mice (CAnN.Cg-Foxn1^NU, 4–6 weeks old). Four weeks after inoculation, the xenograft mice were sacrificed, and at least four implants adhering to the mesothelial surface of each mouse were harvested.

Total RNA was extracted from the metastatic implants of ovarian cancer mouse xenografts and SK-OV-3 cells using the RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions. One microgram of total RNA was converted to cDNA using Superscript II reverse transcriptase and oligo-(dT)_12–18 primers (both from Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. qRT-PCR was performed in a 20 μl reaction mixture containing 1 μl cDNA, 10 μl SYBR Premix EX Taq and 0.4 μl ROX reference dye (50X; both from Takara Bio, Inc. Otsu, Japan), and 200 nM primers for each gene. The primer sequences were: SYTL2 (forward), 5′-GGAGGTCTTCGTATTGGCTTTG-3′; SYTL2 (reverse), 5′-CCATCTTCTCCCAGAGAGCAA-3′; GAPDH (forward), 5′-AATCCCATCACCATCTTCCA-3′; and GAPDH (reverse), 5′-TGGACTCCACGACGTACTCA-3′. The reactions were run on a 7500 Fast Real-Time PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA) at 95°C for 30 sec, followed by 40 cycles of 95°C for 3 sec and 60°C for 30 sec, and a single dissociation cycle of 95°C for 15 sec, 60°C for 60 sec, and 95°C for 15 sec. All PCR reactions were performed in triplicate, and the specificity of the reaction was determined by melting curve analysis at the dissociation stage. Comparative quantification of each target gene was performed based on cycle threshold (CT) normalized to GAPDH using the ΔΔCT method.

Total RNA was extracted from the harvested metastatic implant of ovarian cancer mouse xenografts and SK-OV-3 cells using the RNeasy Mini kit and 1 μg of total RNA was amplified and labeled according to the Affymetrix GeneChip Whole Transcript Sense Target Labeling protocol. The resulting labeled cDNA was hybridized to Affymetrix Human Gene 1.0 ST arrays (Affymetrix, Santa Clara, CA, USA). The scanned raw expression values were background corrected, normalized, and summarized using the Robust Multiarray Averaging approach in the Bioconductor 'affy' package (Affymetrix). The resulting log₂-transformed data were used for further analyses.

To identify differentially expressed genes (DEGs), we applied moderated t-statistics based on an empirical Bayesian approach (10). Significantly upregulated and downregulated DEGs were defined as genes with at least a two-fold difference in expression level between the xenograft cells and the wild-type SK-OV-3 cells after correction for multiple testing [Benjamini-Hochberg-false discovery rate (BH-FDR)-adjusted P-value <0.05] (11).

Finally, we excluded genes with a low expression level (maximum log₂ expression level in a total of eight samples <7.0) from the list of DEGs.

Genomic DNA was extracted from cell lines and tumor tissues using the QIAmp Mini kit (Qiagen), according to the manufacturer's instructions. For analysis of genome-wide screening of DNA methylation, the Illumina HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) was used to target 450,000 specific CpG sites. DNA methylation values were described as β-values, which were calculated by subtracting background using negative controls on the array and taking the ratio of the methylated signal intensity against the sum of both methylated and unmethylated signals. β-values ranged from 0 (completely unmethylated) to 1 (fully methylated) on a continuous scale for each CpG site. To identify differentially methylated CpG sites (DM), we applied the difference in mean β-value (Δβ; mean β-value in tumors - mean β-value in SK-OV-3). If the absolute difference of the mean β-values (|Δβ|) was >0.2, the site was defined as DM. We described a CpG site as hypermethylated if Δβ was >0.2, and hypomethylated if Δβ was <−0.2.

For bisulfite sequencing of the target promoter region of SYTL2, BSP was carried out using conventional PCR in a 50 μl reaction mixture containing 10 ng of bisulfite-modified genomic DNA, 1.5 mM MgCl₂, 200 μM dNTP, and 1 unit of Platinum Taq polymerase (Invitrogen Life Technologies), 1X Platinum Taq buffer and 200 nM of specific BSP forward and reverse primers for each gene. The BSP primers were designed using MethPrimer software (http://www.urogene.org/methprimer). For SYTL2, the BSP product is 345 bp (position in the human GRCh37/hg19 assembly: ch11, 85,437,289~85,437,633) and contained 4 CpG sites. The BSP primer sequences were: (forward), 5′-TTTTTGTTTTATGGGTATTTTTATTTGATA-3′ and (reverse), 5′-AAAAAAACATTACTTTACCAACACTACAAC-3′. The reaction ran at 95°C for 5 min, followed by 30 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec, and a final elongation step at 72°C for 5 min.

BSP products were purified using the QIAquick Gel Extraction kit (Qiagen) according to the manufacturer's instructions and ligated into the yT&A cloning vector (Yeastern Biotech, Taipei, Taiwan). The ligation products were used to transform competent DH5α Esherichia coli cells (RBC Bioscience Corp., Taipei, Taiwan) using standard procedures. Blue/white screening was used to select bacterial clones and BSP product-positive clones were confirmed by the colony PCR using BSP primers to verify insert size. Plasmid DNA was then extracted from at least six insert-positive clones using QIAprep Spin Miniprep kit (Qiagen) and sequenced using M13 primer to analyze methylation status at specific CpG site.

To demethylate methylated CpG sites, SK-OV-3 cells were treated with 0, 5, 10 and 20 μM of 5-aza-dC (Sigma-Aldrich, St. Louis, MO, USA) for 72 h. For co-treatment with 5-aza-dC and TSA, the cells were treated with 10 μM of 5-aza-dC for 72 h and then further treated with 0.5 μM of TSA for 24 h. The media were replaced daily.

To establish a transient expression system, SK-OV-3 and A2780 cells were transfected with pAAV-CBA-SYTL2 or pEGFP-N3 (Clontech, Mountain View, CA, USA) plasmid DNAs using Lipofectamine 2000 (Invitrogen Life Technologies). Briefly, the cells were plated at a density of 5–6×10⁵ cells/well in 6-well plates and allowed to grow overnight. Two micrograms of each plasmid DNA and 5 μl of Lipofectamine 2000 were diluted separately in Opti-MEM medium (Gibco-BRL) to a total volume of 250 μl. The diluted plasmid DNAs and Lipofectamine 2000 were mixed and incubated at room temperature for 20 min to generate the transfection mixtures. The cells were washed with serum-free culture medium, and then the transfection mixtures were added to each well of the 6-well plates containing complete growth medium and incubated at 37°C for 24 h in a 5% CO₂ incubator.

After 24 h of transfection, the transfected cells were starved by serum deprivation. The cell migration assay was performed in 24-well Transwell plates containing inserts with a polycarbonate membrane with an 8.0 μm pore size (Corning Life Sciences, New York, NY, USA). After 24 h of serum deprivation, the cells were detached from the plates and resuspended in serum-free medium at a density of 2–4×10⁶ cells/ml. One hundred microliters of the cell suspension was added to the upper compartment of the Transwell chamber. For each experiment, both chemotactic migration to medium containing 15% FBS and random migration in serum-free medium were assessed in parallel Transwell plates for 6 h at 37°C in a 5% CO₂ incubator.

The in vitro invasion assay was performed using a BD BioCoat Matrigel invasion chamber (Becton-Dickinson, Franklin Lakes, NJ, USA). After 24 h of serum deprivation, the cells were detached from the plates and resuspended in serum-free medium at a density of 1–2×10⁶ cells/ml. One hundred microliters of the cell suspension was added to the upper compartment of the invasion chamber, and 500 μl of culture medium containing 10% FBS was added to the lower compartment of the chamber. The migration through the Matrigel chamber was allowed to proceed at 37°C for 24 h in a 5% CO₂ incubator.

After the incubation period, the cells that had not migrated from the upper side of the filter were carefully scraped away with cotton swabs. The cells on the lower side of the filter were fixed for 2 min using the Diff-Quick kit solution (Thermo Fisher Scientific, Inc., Waltham, MA, USA), stained with 1% crystal violet for 2 min, and washed twice with distilled water at room temperature. The images of the stained cells on the lower side of the membrane were acquired at ×200 magnification in six different fields. For quantitative analysis, the stained cells were subsequently extracted with 10% acetic acid, and colorimetric measurement was performed at 590 nm.

Proteins (30–40 μg) were resolved using denaturing 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% skim milk in Tris-buffered saline with 0.1% Tween-20 and subsequently incubated overnight at 4°C with the following primary antibodies: rabbit anti-SYTL2 polyclonal antibody (1:1,000; Invitrogen), rabbit anti-β-actin monoclonal antibody (1:10,000; Sigma-Aldrich). After washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. Chemiluminescence was detected using WestSave Star substrate (AbFrontier, Seoul, Korea) according to the manufacturer's instructions. Bands were visualized using the Luminescent Image Analyser LAS-300 (General Electric, Chicago, IL USA) and quantified using Image Gauge software (Fujifilm, Valhalla, NY, USA)

The association between the expression level of SYTL2 and prognosis in serous-type ovarian cancer was validated using a recently developed meta-analysis package, curatedOvarianData (12), composed of 2,066 expression profiles and clinical data of ovarian cancer patients from 13 datasets including only serous ovarian cancer. Cox proportional hazards model was used to determine the hazard ratio for overall survival in each dataset and univariate meta-analysis was performed using a fixed effect model for combining hazard ratios.

All data are expressed as mean ± standard deviation of at least three independent experiments. Statistical analyses were carried out using GraphPad Prism5 software (GraphPad, La Jolla, CA, USA) and the details of the statistical analysis for each data set is included in the figure legends. P-values <0.05 were considered to indicate statistically significant results.

Previously, we reported that mRNA expression was altered more than 2-fold in 937 genes, including 444 upregulated genes and 529 downregulated genes, in metastatic implants from ovarian carcinoma xenografts (9). Among the 444 upregulated genes, SYTL2 mRNA expression was upregulated (6.6 to 8.6-fold) in the metastatic implants compared to wild-type SK-OV-3 cells (Fig. 1A). We confirmed overexpression of SYTL2 by qRT-PCR, which showed significantly higher expression (4.3- to 8.5-fold) in all of the implants from the xenografts that we tested (Fig. 1B).

Alteration of DNA methylation has been suggested to lead to aberrant changes in gene expression during cancer progression (13,14). Thus, we performed global DNA methylation profiling of the metastatic tumor implants and wild-type SK-OV-3 cells to determine if DNA methylation changes promoted high expression of the SYTL2 gene during ovarian cancer metastasis. We used the Illumina HumanMethylation450 BeadChip, which covers 25 CpG sites within the promoter region of the SYTL2 gene, which is located at 85,427,572–85,522,637 of chromosome 11 (the human GRCh37/hg19 assembly) (Fig. 2A). Among the 25 promoter CpG sites, 4 CpG sites were differentially methylated (1st, 3rd, 4th and 14th CpGs in Fig. 2A), and three out of the four CpGs were hypomethylated in ovarian metastatic implants compared to the wild-type SK-OV-3 cells (Fig. 2A). Next, we performed BSP of the SYTL2 promoter region on chromosome 11 from 85,437,289–85,437,633 that contained 4 CpG sites in 2 representative tumor tissues (#1 and #3) and in the wild-type SK-OV-3 cell line. The results of BSP showed that the first CpG site was totally unmethylated in all tested samples, whereas the other three CpGs were hypomethylated in the metastatic tumor implants compared to wild-type SK-OV-3 cells (Fig. 2B). Consistent with the Beadchip analysis, DNA methylation of the CpGs at positions 85,437,579 and 85,437,589 was decreased in the metastatic implants compared to SK-OV-3 cells (Fig. 2B).

To determine whether altered mRNA expression of SYTL2 is regulated by epigenetic modification, we treated SK-OV-3 cells with the DNA methyltransferase inhibitor 5-aza-dC and/or the histone deacetylase inhibitor TSA and quantified mRNA expression of SYTL2 by qRT-PCR. After treatment with 5-aza-dC, mRNA expression of SYTL2 was significantly increased (Fig. 3A). A synergic effect on SYTL2 expression was also found after treatment with 5-aza-dC followed by TSA (Fig. 3B). After treatment with 10 μM 5-aza-dC and/or 0.5 μM TSA, decreased methylation status of the SYTL2 promoter was confirmed using BSP (Fig. 3C). These results indicate that SYTL2 gene expression was regulated by a DNA methylation-dependent mechanism.

The SK-OV-3 and A2780 cells were transfected with pAAV-CBA-SYTL2 and pEGFP-N3. The protein level of SYTL2 expression was measured using western blot analysis. The cells transfected by pAAV-CBA-SYTL2 expressed SYTL2 at a much higher level than the cells transfected by pEGFP-N3; ~6.7-fold in SK-OV-3 cells and 4.8-fold in A2780 cells. The data confirmed that SYTL2 was successfully transfected into both human ovarian cancer cell lines (Fig. 4A). To explore the function of SYTL2 on metastasis, we performed in vitro migration and invasion assays using Transwell chambers. We observed a significant increase in migratory ability and invasion activity by overexpression of SYTL2 in both human ovarian cancer cell lines, SK-OV-3 and A2780 cells (Fig. 4B and C). These data suggest that overexpression of SYTL2 conferred migratory and invasive potential to the tumor cells.

To examine the association between the expression level of SYTL2 and overall survival in serous-type ovarian cancer patients, we performed meta-analysis using curatedOvarianData (12), which includes >2,000 expression profiles and clinical data of ovarian cancer patients.

Meta-analysis confirmed that high expression of SYTL2 was significantly associated with poor prognosis in serous-type ovarian cancer patients (Fig. 5). We found that the pooled hazard ratio (HR) estimate was significantly >1 for overall survival, indicating that the risk of death was incerased with increases in SYTL2 expression. More specifically, the combined HR per 1 increment of gene expression was 1.09 (P=0.0048) and the expression change ranged from 3.8 to 14.7 (mean value, 5.8) in 13 datasets. Namely, the pooled HR was estimated at 1.65 by the expression change in the mean value of 5.8.