Research On Abnormal Methylation Of Several Tumor Suppressor Genes In Epithelial Ovarian Carcinoma

Research on Abnormal Methylation of Several Tumor Suppressor Genes in Epithelial Ovarian CarcinomaObjective and significanceCarcinogenesis of epithelial ovarian carcinoma is a stepwise process of accumulation of epigenetic and genetic abnormalities that can lead to cellular dysfunction. Study on abnormal hypermethylation of DNA promoter region is a focus in this area. Epigenetics is defined as modifications of the genome, heritable during cell division, that do not involve a change in the DNA sequence. Epigenetics includes DNA methylation,histone modification,chromatin remodeling and RNA interference, which play important roles in gene expressing regulation. As a main part of epigenetics, DNA hypermethylation is the third way of tumor suppressor gene (TSG) silencing besides genetic gene allelic loss and somatic mutation, which has been associated closely with a wide variety of human cancers. Increasing evidence suggests that many kinds of genes may be hypermethylated in human malignant tumors, including those participate cell cycle control,cell differentiation, apoptosis, metastasis and angiogenesis. DNA methylation and histone acetylation are the two most widely studied epigenetic changes which regulate gene expression. DNA hypomethylation and hyperacetylation can up-regulate gene expression, and DNA hypermethylation and hypoacetylation can down-regulate gene expression. DNA methylation and histone acetylation interplay closely.Ovarian carcinoma is one of the most common gynecologic cancers, and abnormal DNA methylation plays important roles in the course of carcinogenesis and development in epithelial ovarian cancer. Promoter hypermethylation of several TSGs plays crucial roles in carcinogenesis of epithelial ovarian cancer. As suggested, DNA demethlylated agents and histone deacetylase inhibitors can be used in treatment of malignant tumors, including epithelial ovarian carcinoma. Deeply research on hypermethylation of TSGs will not only contribute to further understanding on mechanisms of tumorigenesis, but also promote clinical diagnosis and treatment in epithelial ovarian carcinoma.Materials and methods1 Materials63 surgical resected specimens of sporadic primary epithelial ovarian carcinoma,41 metastatic tissues of pelvic and abdomen cavity,10 adjacent non-cancerous ovarian tissues and 20 normal ovarian tissues were gathered from both Gynecologic Department of the First Affiliated Hospital of China Medical University and Tumor Specific Hospital of Liao Ning Province. Epithelial ovarian cancer lines CAOV3 and OVCaR3 were provided by Cell Biologic Department of China Medical University, HO-8910 by Tumor Research Institute of Shanghai, A2780 by Haematologic Disease Research Institute of Tianjin; Trizol Reagent by GIBCOL BRL company, demethylating agent 5-aza-2'-deoxycitydine(5-Aza-CdR) and histone deacetylase inhibitors(HDACIs) sodium butyrate(NaB) were the product of Sigma; RT-PCR kit was the product of TaKaRa Company; Wizard DNA Clean-up was bought from Promega company, RPMI 1640 culture medium was the product of Hyclone company, RASSF1A goat polyclone,BRCA1 polyclone and P16 mouse monoclone antibody from Santa cruz, hMLH1 rabbit and MGMT mouse monoclone antibody from Lab Vision Neomarkers.2 MethodsMethylation-specific PCP (MSP) was used to detect promoter methylation state. MTT assay, flow cytometry, and Annexin V-FITC staining were performed to analyze the cell proliferation activity, cell cycle, and apoptotic rate. RT-PCR and Western blot were used to detect expression of TSG at mRNA and protein level. Microsatellite instability was detected by PCR- polyacrylamide gel-silver stain method.ResultsSectionⅠPromoter hypermethylation and protein expression of several tumor suppressor genes in epithelial ovarian carcinoma1 Promoter hypermethylation of RASSF1A,BRCA1,p16,hMLH1 and MGMT was detected in epithelial ovarian cancer tissues and metastatic sites, the frequency was 49.2%,25.4%,20.6%,15.9% and 11.1% in ovarian cancer tissues and 58.5%,26.8%,22.0%,12.2% and 9.8% in metastatic sites respectively, which was significantly higher than that in normal ovarian tissues in RASSF1A, BRCA1 and p16 (P＜0.05).2 The frequency of protein expression ofRASSF1A,BRCA1,p16,hMLH1 and MGMT was 31.7%,28.6%,23.8%,27.0% and 44.4% in epithelial ovarian cancer tissues and 29.3%,31.7%,22.0%,24.4% and 48.8% in metastatic sites, which was significantly lower than that in normal ovarian tissues and adjacent non-cancerous ovarian tissues(P＜0.05). The protein expression was related with methylation state of promoter region, which suggested that promoter hypermethylation was one of the reasons that TSG lost expression.3 The frequency of promoter hypermethylation of RASSF1A was significantly lower in epithelial ovarian cancers of stageⅠandⅡthan that in stageⅢandⅣ(P＜0.01), and so was that in both well and moderately differentiated cancers than that in poorly differentiated ones(P＜0.01).4 CIMP was detected in epithelial ovarian carcinoma, and no statistical difference was found between different tumor stages, histological types and differentiated grades when compared with CIMP-negative cancers(P＞0.05). 5 There may exists two different kinds of epithelial ovarian carcinoma, one characterized with hypermethylation in RASSF1A and hMLH1, the other with BRCA1 and p16, maybe including MGMT hypermethylation.SectionⅡMSI and promoter hypermethylation of TSGs in epithelial ovarian carcinoma1 MSI was detected in epithelial ovarian carcinoma, and the frequency of MSI-H was 22.2% and 19.5% in ovarian cancer tissues and metastatic sites, and no statistical difference was found between them(P＞0.05).2 The frequency of MSI-H was significantly higher in mucinous type (38.9%) than that in serous (8.8%) epithelial ovarian cancer(P＜0.05), and so is that in non-serous type(37.9%) (including mucinous, endometrium and clear cell type) than that in serous one (P＜0.01).3 The methylation state of promoter region of hMLH1 and accordant lost expression at protein level correlate with MSI-H(P＜0.01).SectionⅢStudy on regulating effect of synergy of 5-Aza-CdR and NaB on methylation state and expression of TSGs in epithelial ovarian cancer cell lines1 5-Aza-CdR or NaB displayed a growth inhibitory effect on epithelial ovarian cancer cell line CAOV3,OVCaR3,HO-8910 and A2780, and the effect was enhanced when used combinantly(P＜0.05).2 Cell cycle was blocked at G1 phrase after four epithelial ovarian cancer cell lines were exposed to 5-Aza-CdR or NaB, and the effect was enhanced when used combinantly(P＜0.05). 3 The apoptosis rate increased significantly after four epithelial ovarian cancer cell lines were exposed to 5-Aza-CdR or NaB, and the effect was enhanced when used combinantly(P＜0.05).4 After treated with 5-Aza-CdR, TSGs in four epithelial ovarian cancer cell lines displayed different degree of demethylation effect, accompanied by up-regulation of part of TSGs, including p16 in CAOV3,RASSF1A and MGMT in OVCAR3,BRCA1 in A2780, as well as p16 and MGMT in HO-8910 at mRNA and protein level.5 The methylation sate decreased after treated with NaB in RASSF1A in OVCAR3, p16 in CAOV3 and HO-8910, accompanied by up-regulation at mRNA and protein level.6 The methylated TSGs displayed complete demethylation state after treated with 5-Aza-CdR and NaB together, which induced re-exprssion of hMLH1 in A2780 at both mRNA and protein level, up-regulation of RASSF1A in OVCAR3, p16 in HO-8910 and CAOV3.Conclusions1 Promoter hypermethylation of RASSF1A,BRCA1,p16,hMLH1 and MGMT correlates with tumorigenesis and development of epithelial ovarian carcinoma.2 MSI exists in epithelial ovarian carcinoma, and MSI-H correlates with tumorigenesis and development of non-serous type epithelial ovarian carcinoma, especially mucinous type. The mechanism of MSI-H in epithelial ovarian carcinoma lies partly in promoter hypermethylation of hMLH1 gene.3 5-Aza-CdR can reverse the abnormal hypermethylation state of RASSF1A,BRCA1,p16,hMLH1 and MGMT in human epithelial ovarian cancer cell lines and regulate the expression of some of the TSGs. 4 NaB coordinate with 5-Aza-CdR in inducing demethylation and regulating gene expression.5 5-Aza-CdR and NaB can inhibit the growth of epithelial ovarian cancer cells synergistically, which may be due to blocking cell cycle,inducing apoptosis and up-regulation of RASSF1A,p16 and BRCA1.