Yonsei Med J.  2014 Sep;55(5):1206-1213. 10.3349/ymj.2014.55.5.1206.

Overexpression of Mucin 13 due to Promoter Methylation Promotes Aggressive Behavior in Ovarian Cancer Cells

• Sung HY
• Park AK
• Ju W
• Ahn JH

• KMID: 1799482
• DOI: http://doi.org/10.3349/ymj.2014.55.5.1206

Abstract

PURPOSE
Recent discoveries suggest that aberrant DNA methylation provides cancer cells with advanced metastatic properties. However, the precise regulatory mechanisms controlling metastasis genes and their role in metastatic transformation are largely unknown. To address epigenetically-regulated gene products involved in ovarian cancer metastasis, we examined the mechanisms regulating mucin 13 (MUC13) expression and its influence on aggressive behaviors of ovarian malignancies.
MATERIALS AND METHODS
We injected SK-OV-3 ovarian cancer cells peritoneally into nude mice to mimic human ovarian tumor metastasis. Overexpression of MUC13 mRNA was detected in metastatic implants from the xenografts by expression microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The DNA methylation status within the MUC13 promoter region was determined using bisulfite sequencing PCR and quantitative methylation-specific PCR. We evaluated the effects of exogenous MUC13 on cell invasion and migration using in vitro transwell assays.
RESULTS
MUC13 mRNA expression was up-regulated, and methylation of specific CpG sites within the promoter was reduced in the metastatic implants relative to those in wild-type SK-OV-3 cells. Addition of a DNA methyltransferase inhibitor to SK-OV-3 cells induced MUC13 expression, thereby implying epigenetic regulation of MUC13 by promoter methylation. MUC13 overexpression increased migration and invasiveness, compared to control cells, suggesting aberrant up-regulation of MUC13 is strongly associated with progression of aggressive behaviors in ovarian cancer.
CONCLUSION
We provide novel evidence for epigenetic regulation of MUC13 in ovarian cancer. We suggest that the DNA methylation status within the MUC13 promoter region may be a potential biomarker of aggressive behavior in ovarian cancer.

Keyword

Ovarian cancer; mouse xenograft; MUC13; DNA methylation

MeSH Terms

Animals
Cell Line, Tumor
*DNA Methylation
Epigenesis, Genetic
Female
*Gene Expression Regulation, Neoplastic
Heterografts/metabolism
Humans
Mice
Mice, Nude
Mucins/*genetics/metabolism/physiology
Neoplasm Invasiveness/genetics
Ovarian Neoplasms/genetics/*metabolism/pathology
RNA, Messenger/metabolism
Mucins
RNA, Messenger

Figure

• 

  Fig. 1 MUC13 expression is up-regulated in metastatic implants from xenograft mice. MUC13 mRNA expression was measured by expression microarray (A) and qRT-PCR (B). The error bars indicate means±standard deviation of triplicate experiments. The metastatic implants from each mouse xenograft are labeled 1C-8C (n=7). qRT-PCR, quantitative reverse-transcription polymerase chain reaction.

• 

  Fig. 2 DNA methylation is altered at CpG sites in the MUC13 promoter in metastatic implants from mouse xenografts. The DNA methylation status was analyzed using Bisulfite sequencing analysis (A). The MUC13 promoter region is located at positions 124653578-124653990 in the human GRCh37/hg19 assembly and contains six CpG residues within chromosome 3. The six CpGs are located at positions -273, -159, -123, -64, -37, and -21 from the transcription start site. Each circle represents CpG dinucleotides. The methylation status of each CpG site is illustrated by black (methylated) and white (unmethylated) circles, and the total percentage of methylation at each site is indicated by a pie graph on the bottom line. The black segment of the pie graph indicates the methylated CpG percentage, whereas the white segment represents the unmethylated CpG percentage. The DNA methylation status at the -64 CpG site was analyzed using qMSP (B). Triangles above the circles in A indicate the specific CpG site used for qMSP. Data are shown as the means±SD (n=3). Statistical analyses were performed using a one-way ANOVA and subsequent Bonferroni tests (*p<0.05). M, the percentage of methylated CpGs; U, the percentage of unmethylated CpGs; qMSP, quantitative methylation-specific PCR; PCR, polymerase chain reaction; ANOVA, analysis of variance.

• 

  Fig. 3 MUC13 expression changes following demethylation in SK-OV-3 cells. The SK-OV-3 cells were treated for 3 days with 0, 5, and 10 µM 5-aza-2'-deoxycytidine, respectively. After treatment with 5-aza-2'-deoxycytidine, the DNA methylation status at the -64 CpG site of MUC13 was analyzed using qMSP (A), and MUC13 mRNA expression was measured by qRT-PCR (B). Data are shown as the means±SD (n=3). Statistical analyses were performed using a one-way ANOVA and subsequent Bonferroni tests (*p<0.05). M, the percentage of methylated CpG; U, the percentage of unmethylated CpG; qMSP, quantitative methylation-specific PCR; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; ANOVA, analysis of variance.

• 

  Fig. 4 MUC13 promotes migration and invasiveness of SK-OV-3 cells. Migration of serum-starved cells towards 15% serum-containing medium was determined by the transwell assay. Cells that migrated through an 8-µm pore filter were fixed and stained with crystal violet. Representative images of migrated cells transfected with EGFP or MUC13 are shown (A). Quantitative analysis of migrated cells was carried out by measuring the absorbance of extracts of cell stains at 595 nm. Data are shown as means±SD for triplicate measurements (B). Statistical analysis was performed using a t-test (*p<0.05). Invasion by serum-starved cells towards 10% serum-containing medium was determined using a Matrigel-coated invasion chamber. The cells invading through the Matrigel were fixed and stained with crystal violet. Representative images of invading cells transfected with EGFP or MUC13 are shown (C). Quantitative analysis of invading cells was carried out by measuring the absorbance of extracts of cell stains at 595 nm. Data are shown as means±SD for triplicate measurements (D). Statistical analysis was performed using a t-test (*p<0.05). EGFP, enhanced green fluorescent protein.

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