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Objective To investigate the effect of 5-aza-2'deoxycytidine (5-Aza-CdR) on proliferation and expression of RASSF1 A gene in human ovarian cancer cell line SKOV3 and 3AO.Methods SKOV3 and 3AO cells were treated with different concentrations (0.5,5,50 μmol/L) of DNA methyltransferase inhibitor 5-Aza-CdR.RT-PCR and Western Blot were adopted to detect expression of mRNA and protein of RASSF1A gene before and after treatment with 5-Aza-CdR respectively.Results Compared with control group,when the 5-Aza-CdR concentration was 0.5,5,50 μmol/L after drug treatment,human ovarian cancer cells could significantly inhibit tumor cell growth; SKOV3 and 3AO cells in control group were observed weaker expression of RASSF1A mRNA.After treated with 5-Aza-CdR,the expressions of RASSF1A mRNA were observed increased with the increase of the drug concentration.After treated with different concentration of 5-Aza-CdR,the expressions of RASSF1A mRNA treated with 0.5 μmol/L 5-Aza-cdR was lower than those treated with 5 and 50 μmol/L 5-Aza-cdR (t =-8.866,P =0.01 ; t =-12.256,P =0.000).However,expressions of RASSF1A mRNA treated with 5 and 50 μmol/L 5-Aza-cdR respectively showed no statistical significance (t =0.431,P =0.689).Expressions of RASSF1A protein treated with 0.5 μmol/L 5-Aza-cdR and 5 μmol/L 5-Aza-cdR didn't show statistically significant (t =-1.586,P =0.188).Conclusion Expressions of RASSF1A mRNA and protein in SKOV3 and 3AO cells were evidently enhanced.As one kind of methyltransferases inhibitors,5-Aza-CdR can inhibit ovarian cancer cell line SKOV3,3AO growth through the RASSF1A promoter methylation,and thus promote their apoptosis.
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Objective To access the role of methylated RUNX3 ggene in the carcinogenesis an progression of ovarian carcinoma.Methods Sample of 32 epithelial ovarian carcinoma tissues,32 para-carcinoma tissues,36 benign epithelial tumors and 10 normal ovarian tissues and 2 cell lines(3A0 and SKOV3),were collected and subject to methylation-specific PCR(MSP).Promoter methylation status of RUNX3 in two tumor cell lines were analyzed before and after 5-aza-2'deoxycytidine treatment.In addition. mRNA expression of RUNX3 were investigated by quantitative reverse transcription-PCR Results CpG island methylation of RUNX3 was observed in 53.1%(17 of 32)of epithelial ovarian cancer,and 37.5% (12 of 32)in corresponding noncancerous tissues,16.7% in benign epithelial tumors(6 of 36),and all celllines,but not in normal control tissues.The prevalence of RUNX3 gene CpG methylation in malignant was significantly higher than those in benign and normal tissues(X2=10.060,X2=8.925,P<0.05). Nineteen percent(6 of 32)of ovarian epithelial carcinoma expressed RUNX3 mRNA,while its expression Was present in 28% (9 of 32)corresponding noncancerous tissues and 72% (26 of 36)of benign ovarian tumor and 80% (8 of 10)of normal ovarian tissues.The RUNX3 promoter methylation was found in all cell lines tested.The ratio of expression of RUNX3 mRNA in ovarian epithelial carcinoma was significantly lower than those of normal and benign tumors(X2=19.443,X2=12.862,P<0.05).After 5-aza-2'deoxycytidine treatment,methylation was partially or completely reversed,and its mRNA expression was increased.The relationship between gene expression and promoter methylation was reversely correlated.Conclusions Our results suggest that promoter hypermethylation of RUNX3 genes is common in ovarian cancer. Therefore, hypermethylation of RUNX3 genes may be involved in the carcinogenesis of ovarian cancer and may serve as an early diagnostic marker for ovarian cancer. The close correlation between RUNX3 methylation of its mRNA suggests that methylation which can be reversed.Thus,it provides a new way for therapy of ovarian Cancer.
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Objective To observe the effects of 5-Aza-2’-deoxycytidine(5-Aza-CdR)on cell proliferation and apoptosis in ovarian cancer cell line SKOV3 and the expression of mismatch repair gene HMLH1/HMSH2, and to investigate the potential mechanism of its antitumorigenesis. Methods Human ovarian cancer cell line SKOV3 was treated with 5-Aza-CdR(0.5, 5 and 50 ?mol/L), a specific demethylation agent for 3 d, and then cultured in RPMI-1640 medium for 7 d.The growth of cells was examined by MTT assay. The apoptosis was analyzed by flow cytometry. The expression of HMLH1 and HMSH2 mRNA was observed by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR). Results SKOV3 cells treated with 5-Aza-CdR displayed a slow growth rate in comparison with that of the control cells, The apoptosis rates of each group were 10.59%?1.57%、17.52%?1.72%、34.10%?1.45%,respectively,which were markedly higher than that of control(P