5-氮-2′脱氧胞苷对人卵巢癌细胞RUNX3基因启动子区甲基化状态的影响

Methylation status of promoter of RUNX3 gene in epithelial ovarian cancer cell lines

目的研究抑癌基因RUNX3启动子区在人卵巢癌细胞系(3AO、SKOV3)及正常卵巢细胞系(NOEC)中的甲基化状态,以及5-氮-2′脱氧胞苷(5-aza-2′-deoxycytidine)对人卵巢癌细胞3AO及SKOV3增殖与凋亡及RUNX3 mR-NA表达的影响,探讨RUNX3基因甲基化与卵巢癌发生及发展的关系。方法用甲基化特异性PCR(MSP)法检测经特异性甲基转移酶抑制剂5-氮-2′脱氧胞苷处理前后卵巢癌细胞3AO、SKOV3以及NOEC中RUNX3基因启动子区的甲基化状态;并用0.5、5、50μmol/L5-氮-2′脱氧胞苷处理人卵巢癌细胞株3AO及SKOV3共3d,继续常规培养5d后,采用四唑盐(MTT)比色观察细胞经药物处理前后的生长活性,以半定量RT-PCR检测细胞经药物处理前后抑癌基因RUNX3mRNA的表达,应用流式细胞仪进行细胞凋亡率的检测。结果正常卵巢细胞中未见RUNX3启动子甲基化,卵巢癌细胞3AO及SKOV3均发现RUNX3启动子甲基化现象;卵巢癌细胞3AO、SKOV3均可见RUNX3mRNA表达,但与正常卵巢细胞比较表达较弱,经药物处理癌细胞后其RUNX3mRNA的表达较前明显增强,且其表达强度与5-氮-2′脱氧胞苷的浓度存在剂量依赖关系;与加药前比较,5-氮-2′脱氧胞苷在0.5、5、50μmol/L浓度时均能明显抑制肿瘤细胞生长,随5-氮-2′脱氧胞苷浓度增加,细胞生长速率下降,加药前细胞凋亡率SKOV3为(2.71±0.81)%;3AO为(3.23±0.68)%,经5-氮-2′脱氧胞苷0.5、5、50μmol/L处理细胞后,细胞凋亡率SKOV3为(2.71±0.81)%、(15.43±1.33)%、(25.55±1.61)%;3AO为(10.66±2.09)%、(16.51±1.42)%、(25.46±1.55)%。与加药前比较,差异均有统计学意义(P<0.01),且凋亡率与5-氮-2′脱氧胞苷浓度呈剂量依赖关系(F=138.7;142.3,均P<0.05)。结论特异性甲基转移酶抑制剂5-氮-2′脱氧胞苷可逆转、恢复并增强人卵巢癌细胞系3AO及SKOV3中RUNX3基因表达,抑制癌细胞生长及诱导部分癌细胞凋亡。

Objective To investigate the methylation status of RUNX3 promoter region in the epithelial ovarian cancer cell line and effect of 5-aza-2＇-deoxycytidine on the proliferation and apoptosis of ovarian cancer cells and the expression of tumor suppressor gene RUNX3. Methods Methylation status of RUNX3 promoter region was assayed in human ovarian cancer cell line 3AO, KOV3 and normal ovarian epithelial cell （ NOEC ） by methylation -specific PCR（MSP）. Ovarian cancer cell line was treated with 5-aza- 2＇-deoxycytidine （0. 5,5,50 μmol/L） , a specific demetbylating agent for 3 d, and then cultured in RPMI 1640 medium for 5 d. The growth of 3AO and SKOV3 cells was observed by MTI＂ assay before and after 5- aza-2＇-deoxycytidine treatment respectively. The expression of RUNX3 mRNA was observed by semi- quantitative reverse transeription-polymerase chain reaction （RT-PCR）. The apoptosis of 3AO and SKOV3 cells was analyzed by flow cytometry. Results The methylation of RUNX3 promoter was found in both 3AO and SKOV3 but not in NOEC. Ovarian cancer cells treated with 5-aza-2＇-deoxycytidine displayed a slowed growth in comparison with the control cells, and methylation was induced or completely reversed and their mRNA expression was increased differently. Conclusions 5-aza-2＇-deoxyeytidine as a methylase inhibitor can reverse, recovery and reinforce RUNX3 mRNA expression of ovarian cell line 3AO and SKOV3, repress cancer cell growth and induce its apoptosis.