5-氮杂-2-脱氧胞苷对母系表达基因3启动子超甲基化的逆转作用及其对卵巢癌细胞增殖活性的抑制作用  

Reversal effect of 5-aza-2-deoxycytidine on the maternally expressed gene 3 promoter hypermethylation and its inhibitory effect on the proliferation of epithelial ovarian cancer cells

作　　者：李剑琦[1] 周冬梅[1] 汪志辉[1] 谭琳玉 周艳清[1] 李娟[1] 生秀杰[1] 

机构地区：[1]广州医科大学附属第三医院妇科,广东省产科重大疾病重点实验室,广东省普通高校生殖与遗传重点实验室,510150

出　　处：《中华肿瘤杂志》2015年第5期324-329,共6页Chinese Journal of Oncology

基　　金：广东省科技厅基金（2013B021800307）

摘　　要：目的探讨不同浓度DNA甲基化抑制剂5-氮杂。2．脱氧胞苷对母系表达基因3（MEG3）基因启动子超甲基化的逆转作用，以及恢复MEG3表达对上皮性卵巢癌细胞增殖活性的影响。方法以0、1、5、10、20μmol／L的5-氮杂-2-脱氧胞苷作用上皮性卵巢癌OVCAR3细胞6d后，采用甲基化特异性PCR（MSP）检测MEG3启动子的甲基化状态，逆转录聚合酶链反应（RT-PCR）检测MEG3mRNA的表达水平，采用四甲基偶氮唑蓝（MrIT）比色法和5-乙炔基-2-脱氧尿嘧啶核苷（EdU）掺人实验检测细胞增殖活性的变化。结果MSP检测显示，0μmol／L5-氮杂-2-脱氧胞苷组（对照组）、1p,mol／L5-氮杂-2一脱氧胞苷组、5μmol／L5-氮杂-2-脱氧胞苷组、10μmoL／L5-氮杂-2-脱氧胞苷组和20μmol／L5-氮杂-2-脱氧胞苷组的甲基化水平分别为1．00±0．00、0．79±0．00、0．67±0．00、0．65±0．03和0．61±0．01，各组间差异均有统计学意义（均P〈0．05）。RT-PCR检测显示，对照组、1μmol／L5-氮杂-2-脱氧胞苷组、5IxmoL／L5-氮杂-2-脱氧胞苷组、10μmol／L5-氮杂-2-脱氧胞苷组和20μmol／L5-氮杂-2-脱氧胞苷组MEG3mRNA的相对表达水平分别为1．00±0．00、2．04±0．16、2．44±0．17、3．19±0．34和5．34±0．39，各组间差异均有统计学意义（均P〈0．05）。MTT比色法检测显示，与对照组比较，1μmol／L5-氮杂-2-脱氧胞苷组、5μmol／L5-氮杂-2-脱氧胞苷组、10μmol／L5-氮杂-2-脱氧胞苷组和20μmol／L5-氮杂-2-脱氧胞苷组OVCAR3细胞的增殖活性受到明显抑制，5-氮杂-2-脱氧胞苷作用2、4、6d时，各组间细胞抑制率差异均有统计学意义（均P〈0．01）。EdU掺人实验显示，对照组、1p,mol／L5-氮杂-2一脱氧胞苷组、5μmoL／L5-氮杂-2-脱氧胞苷组、10μmol／L5-氮杂-2-脱氧胞苷组和20μmol／L5-氮杂-2-脱氧胞苷组OVCAR3细胞中的EdU阳性细胞率分别为（40．78＋0．80）％、（35．65±0．33）�Objective To investigate the reversal effects of different concentrations of DNA methylation inhibitor, 5-aza-2-deoxycytidine, on the hypermethylation of maternally expressed gene 3 （MEG3） gene promoter, and then the inhibitory effect of restoration of MEG3 expression on the proliferation of ovarian cancer cells. Methods Human ovarian cancer OVCAR3 cells were treated with various concentration of 5-aza-2-deoxycytidine （0, 1, 5, 10, 20 μmol/L, respectively） for 6 days. Then the methylation status of MEG3 promoter was detected by methylation specific PCR （MSP）. The alteration of MEG3 gene expression was detected by RT-PCR. Cell proliferation was determined by MTY assay and EdU incorporation assay. Results After treated with 5-aza-2-deoxycytidine, the methylation status of MEG3 in the 0, 1, 5, 10, 20 μmol/L 5-aza-2-deoxycytidine groups were 1.00±0.00, 0.79±0.00, 0.67v0.00, 0.65± 0.03 and 0.61±0.01 folds, respectively （P〈0.05 for all）. The relative expressions of MEG3 mRNA in the 0, 1, 5, 10, 20 μmol/L 5-aza-2-deoxycytidine groups were 1.00±0.00, 2.04±0.16, 2.44±0.17, 3.19±0.34 and 5.34±0.39, respectively （P〈0.05 for all）. In contrast to the negative control, the inhibition rates of the OVCAR3 cell growth were increased significantly when treated with 1, 5, 10, 20 μmol/L 5-aza-2- deoxycytidine in 2, 4 and 6 days. There were （ 40.78 ± 0.80） %, （ 35.65 ± 0.33 ） %, （ 31.81± 0.66 ） %, （27.33±1.27）% and （17.75±1.85）% of EdU-positive cells in the 0, 1, 5, 10 and 20 Ixmol/L 5-aza-2- deoxycytidine groups （P 〈 0.01 for all ）. Conclusions Maternally expressed gene 3 promoter hypermethylation is reversed by 5-aza-2-deoxycytidine in ovarian cancer ceils. The downregulation of MEG3 gene might be resulted from the methylation, and the re-expression of MEG3 partly contribute to the growth inhibition of epithelial ovarian cancer cells.

关 键 词：卵巢肿瘤 甲基化 细胞增殖 母系表达基因3 5-氮杂-2-脱氧胞苷 

分 类 号：R737.31[医药卫生—肿瘤]