Abstract
Objective In the current study, the role of abnormal methylation of Wnt5a gene promoter regions in human epithelial ovarian cancer was investigated. Methods Wnt5a expressions were examined by immunohistochemistry in epithelial ovarian tissues (30 normal and 79 human EOC tissues). SKOV3 cells were treated with different concentrations of 5-Aza-CdR (0.5, 5, and 50 μmol/L). The methylation status of the Wnt5a promoter was analyzed using a methylation-specific polymerase chain reaction (MSP), and the expression level of Wnt5a mRNA was detected using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was measured by MTT assay, and apoptosis was analyzed using flow cytometry. Results (1) Compared with normal tissues, Wnt5a expressions were reduced or lost in EOC (P < 0.05). Wnt5a expression had a close relationship with histological grade, FIGO stage, and lymph node metastasis (P = 0.005, P = 0.022, and P = 0.037, resp.). (2) Wnt5a abnormal methylation status existed in ovarian cancer tissues and was higher than that of normal ovarian tissue (P < 0.01). (3) Before treatment with 5-Aza-CdR, the promoter of the Wnt5a gene was methylated in SKOV3 cells; accordingly, Wnt5a mRNA levels were low to absent in SKOV3 cells. (4) Following 5-Aza-CdR treatment, MSP analysis revealed complete demethylation of the Wnt5a promoter in the SKOV3 cell line, particularly at 5 μmol/L 5-Aza-CdR. Wnt5a expression increased in SKOV3 cells following treatment with a demethylating agent (P ≤ 0.001). (5) The growth rate of the cells was inhibited in a dose-dependent manner by treatment with 5-Aza-CdR. (6) The cell apoptosis rate increased gradually after treatment with 0.5, 5, and 50 μmol/L 5-Aza-CdR. The apoptosis rate exists in a dose-dependent relationship with 5-Aza-CdR concentration (F = 779.73, P < 0.01). Conclusions Wnt5a gene region promoter aberrant methylation existed in epithelial ovarian cancer, and abnormal methylation of Wnt5a gene promoter regions may be a new target for the treatment of epithelial ovarian cancer.
Highlights
The characteristics of ovarian cancer include a difficult early diagnosis, rapid development, and high mortality
methylation-specific polymerase chain reaction (MSP) was used for the analysis of the Wnt5a methylation status (Figure 2). 31 of 79 (39.24%) methylation were tested in epithelial ovarian cancer (EOC), while only 3 of 30 (10%) was found in normal ovarian tissues
CpG dinucleotides are the major site of DNA methylation, which is nonrandomly distributed throughout the genome
Summary
The characteristics of ovarian cancer include a difficult early diagnosis, rapid development, and high mortality. More than 70% of ovarian cancer patients are diagnosed at an advanced stage, and the five-year survival rate is only 20%. As a result of in-depth studies in recent years, abnormalities of epigenetic modifications have been found to be one of the important reasons for tumor formation, are involved in the occurrence and development process of tumors, and are closely related to some pathologic types and prognosis. Epigenetic modification includes DNA methylation, microRNA regulation abnormalities, and histone acetylation. DNA methylation is one of the most important methods of epigenetic regulation, which can cause changes to chromatin structure, DNA conformation, DNA stability, DNA-protein interaction, and gene expression.
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