Study On Wnt5a Expression And Its Promoter Regions Methylation Status In Epithelial Ovarian Cancer

Objective:The purpose of this study is to explore the correlation between the expression of Wnt5a protein and promoter regions methylation status of Wnt5a in epithelial ovarian tumors; the effect of5-Aza-2’-deoxycytidine (5-Aza-CdR) on the hypermethylation of the Wnt5a gene promoter and impact on cell proliferation was examined in the human epithelial ovarian cancer cell line, SKOV3, and the expression level of Wnt5a mRNA. To provide a theoretical basis for Wnt5a as the diagnosis of epithelial ovarian cancernew tumor marker and new therapeutic targets.Method:1. Immunohistochemical method was used to assess the expression of Wnt5a in20cases of normal ovarian tissues,16cases of benign ovarian tumors,18cases of borderline ovarian tumors and79cases of epithelial ovarian cancer.2. Methylation status of the Wnt5a promoter regions was studied using methylation-specific PCR (MSP)(30normal.79human epithelial ovarian cancer tissues and the human epithelial ovarian cancer cell line, SKOV3).3. MTT assay different concentrations of5-Aza-CdR (0.5.5,50μmol/L) intervention in human epithelial ovarian cancer cell line SKOV3proliferation inhibition, to determine the appropriate drug concentration and time of the post-experiment;The applications status of SKOV3cells were treated with different concentrations of5-Aza-CdR (0.5,5, and50μmol/L) was analyzed by flow cytometric.SKOV3cells were treated with different concentrations of5-Aza-CdR (0.5,5, and50μmol/L);The methylation status of the Wnt5a promoter was analyzed using methylation-specific polymerase chain reaction (MSP), and the expression of Wnt5a mRNA was detected by real-time fluorescence quantitative PCR.Results:1. The expression of Wnt5a protein in epithelial ovarian cancer was significantly lower than that in normal ovarian, and benign ovarian (P<0.01); The expression of Wnt5a protein was closely related with histopathological grading (P<0.01). clinical stage (P<0.01) and lymph node metastasis (P<0.05).2.(1) Wnt5a promoter region methylation level was significantly higher than normal ovarian tissue, the difference was statistically significant (P<0.01):combined with Wnt5a protein expression, in epithelial ovarian epithelial ovarian cancer they were negatively correlated γ=-0.713, P<0.01:39.24%of carcinomas were methylated, while10%of normal tissues were methylated. The expression of Wnt5a promoter regions hypermethylation was closely related with histopathological grading (P<0.05), clinical stage (P<0.05) and lymph node metastasis (P<0.05).(2) Wnt5a promoter regions were methylated in SKOV3cells.3. Different concentrations of5-Aza-CdR treatment SKOV3cells, with the increase in the concentration of inhibition of5-Aza-CdR enhanced, and the suitable time for SKOV3cells treat with5-Aza-CdR was48h.SK.OV3cells apoptosis rate for intervention of5-Aza-CdR was statistically significant, the apoptosis rate with5-Aza-CdR concentration has a dose-dependent manner (F=214.54, P <0.01).SKOV3cells treat with5-Aza-CdR, Wnt5a gene promoter region methylation-specific band disappeared, the unmethylated specific bands enhanced. Without5-Aza-CdR treatment SKOV3cells as a control group Wnt5a gene mRNA showed low expression or non-expression, SKOV3cells were treated with different concentrations of5-Aza-CdR treatment Wnt5a mRNA expression increased or re-expression of5μmol/L,50μmol/L group compared with control group (P<0.05) the difference was statistically;5μmol/L group and50μmol/L, the pairwise comparison of the difference was statistically significant (P<0.01).Conclusion:1. Wnt5a protein in normal ovarian tissue, benign ovarian tumor, borderline ovarian tumor tissue and epithelial ovarian cancer tissue expression were decreased, this conclusion was shown that the expression decreased or deleted of Wnt5a protein may be associated with the incidence of epithelial ovarian cancer; Wnt5a protein expression in epithelial ovarian cancer was associated with pathological grade, clinical stage, lymph node metastasis, Wnt5a protein expression in ovarian cancer patients indicate that the clinical outcome may be better.2. Wnt5a promoter region of epithelial ovarian cancer tissues appeared aberrant methylation, with Wnt5a protein exist negative correlation, which may be one of the important reasons for Wnt5a protein expression decreased or deleted.3.5-Aza-CdR can effectively reverse the methylation status of Wnt5a promoter regions in SKOV3cells, which made Wnt5a gene re-expressed, and suppresses SKOV3cells growth and promotes apoptosis.