A Study On Methylation Of Mismatch Repair Genes HMLH1 And HMSH2 In The Development And Progression Of Ovarian Carcinoma

Part one Methylation status of promoter of mismatch repair genes hMLH1 and hMSH2 in epithelial ovarian carcinoma[Objective] To explore the methylation status of DNA mismatch repair(MMR) genes hMLH1 and hMSH2 promoter region in the epithelial ovarian carcinoma and its role in oncogenesis, and to investigate the relationship between methylation of DNA MMR genes and diagnose and /(or)prognosis of ovarian carcinoma, to evaluate the possibility of them serving as predictive/prognostic parameter.[Methods] Methylation status of hMLH1 and hMSH2 promoter region wasassayed in 20 normal ovarian tissues, 25 benign epithelial tumor, 56 malignant epithelial tumor and cell lines SKOV3,3AO by methylation-specific polymerase chain reaction(MSP). SKOV3 and 3AO were analyzed before and after 5-aza-2'-deoxycytidine (5-Aza-CdR) treatment. In addition, alteration of mRNA expression of hMLH1 and hMSH2 was observed by reverse transcription polymerase chain reaction (PT-PCR). [Results] No methylation of hMLH1 and hMSH2 promoter was found innormal ovarian tissues. CPG islands methylation of hMLH1 and hMSH2 was observed in 4.0% (1/25 ),8.0% (2/25) respectively in benign epithelial tumor, 30.4% (17/56),51.8% (29/56) respectively in malignant epithelial tumor. Methylation status in promoter showed obvious correlation with pathological grade and lymph node metastasis (P<0.05). After 5-Aza-CdR (0.5μm/L) treatment, hMLH1 and hMSH2 methylation was induced or completely reversed, and their mRNA expression was increased differently.[Conclusions] Methylation of hMLH1 and hMSH2 may involved in thecarcinogenesis of ovarian carcinoma and may serve as predictive/prognostic parameter. The close correlation between hMLH1 and hMSH2 methylation of their mRNA suggests that methylation which can be reversed, is an important pathway in the regulation of gene expression. Part twoEffect of 5-aza-2'-deoxycytidine on cell proliferation and apoptosis in ovarian carimoma cells and on expression of mismatch repair genes hMLH1 and hMSH2[Objective] To observe the expression of mismatch repair(MMR) geneshMLHl and hMSH2 in ovarian carcinoma cell line SKOV3 and 3AO; To investigate the effects of 5-aza-2'-deoxycytidine (5-Aza-CdR) on cell proliferation and apoptosis in SKOV3 and 3AO and expression of hMLH1 and hMSH2, thus, to seek a new way for therapy of ovarian carcinoma.[Methods] Human ovarian carcinoma cell line SKOV3 and 3AO was treatedwith 5-Aza-CdR(0.5,5, 50μmol/L), a specific demethylation agent for 3d, and then cultured in RPMI-1640 medium for 7d.The growth of cells was observed by MTT assay before and after 5-Aza-CdR treatment, respectively. The apoptosis of cells was analyzed by flow cytometry (FCM) . The expression of hMLH1 and hMSH2 mRNA was observed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).[Results] SKOV3 and 3AO cells treated with 5-Aza-CdR(0.5, 5,50μmol/L) displayed a slowed growth rate in comparison with the control cells, and the growth rate decreased accordingly with the increase of 5-Aza-CdR concentration. The apoptosis rate of each group for SKOV3 were (10.59±1.57)%,(17.52±1.72)%, (34.10±1.45)%, respectively , which were markedly higher than (5.35±0.86)% of control (P<0.01) . The apoptosis rate of each group for 3AO were (11.11±2.21)%, (17.24±1.11)%, (26.53±2.00)% , respectively , which were markedly higher than(3.39±0.71)% of control (P<0.01) . We also found that the apoptosis rate was positively correlated with 5-Aza-CdR concentration (F_SKOV3=227.6, P_SKOV3<0.01; F_3AO=108.4, P_3AO<0.01) . After 5-Aza-CdR treatment, the level of hMLH1 and hMSH2 mRNA expression was increased differently, and it was significantly correlated with the concentration of 5-Aza-CdR.[Conclusions] In human ovarian carcinoma cell line SKOV3 and 3AO ,5-Aza-CdR can make the deactivated MMR genes hMLH1 and hMSH2 re-expression, inhibit the proliferation and partly induce the apoptosis.