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目的地西他滨(decitabine,DAC)具有较好的抗肿瘤活性,其分子机制与DNA去甲基化有关,本研究通过DAC对腺瘤性结肠息肉病(adenomatous polyposiscoli,APC)基因的表达水平和去甲基化的影响,探讨DAC诱导宫颈腺癌Hela细胞凋亡分子机制。方法不同浓度的DAC作用于宫颈腺癌Hela细胞24、36和48h,MTT法检测对宫颈癌细胞的增殖抑制作用,观察DAC对Hela细胞增殖的浓度依赖效应和时间依赖效应。流式细胞术检测DAC对宫颈腺癌Hela细胞凋亡和细胞周期的影响。在DAC作用于宫颈腺癌Hela细胞前后,通过甲基化特异性PCR(Methylation specific PCR,MSP)检测APC基因的甲基化状态;RT-PCR法检测APC基因mRNA表达水平;蛋白质印迹法检测APC蛋白、β-catenin蛋白在胞内及核内表达的变化。结果 DAC对宫颈腺癌Hela细胞增殖抑制具有浓度依赖效应和时间依赖效应,Hela细胞半数抑制浓度(IC50)24、36、48h分别为28.23、7.65和5.64μmol/L。DAC处理后的宫颈腺癌Hela细胞的凋亡率为(73.82±0.11)%,明显高于对照组的(12.41±0.24)%,P<0.001。DAC处理Hela细胞后,S期细胞比例(47.82±2.57)%和G2期细胞比例(30.87±2.28)%显著高于空白对照组的S期细胞比例(24.08±0.71)%和G2期细胞比例(2.52±0.84)%,P<0.001。DAC处理后,APC基因启动子区域去甲基化状态明显增高,APC mRNA表达量上升,处理前后比较差异有统计学意义,P<0.05。DAC处理后,胞内APC蛋白表达上调,而胞内和核内的β-catenin蛋白表达下调,差异均有统计学意义,P<0.05。结论 DAC可通过对APC基因的去甲基化作用,上调胞内APC蛋白表达,下调胞内和核内β-catenin表达,诱导宫颈癌细胞凋亡。
The target decitabine (DAC) has good anti-tumor activity, and its molecular mechanism is related to demethylation of DNA. In this study, DAC was used to determine the expression level of adenomatous polyposiscoli (APC) And demethylation, to investigate the molecular mechanism of DAC-induced cervical adenocarcinoma Hela cell apoptosis. Methods Different concentrations of DAC were applied to cervical adenocarcinoma Hela cells for 24, 36 and 48 h. MTT assay was used to detect the proliferation of cervical cancer cells. The concentration-dependent and time-dependent effects of DAC on Hela cell proliferation were observed. Flow cytometry was used to detect the effect of DAC on the apoptosis and cell cycle of cervical adenocarcinoma Hela cells. The methylation status of APC gene was detected by methylation specific PCR (MSP) before and after DAC treatment on cervical adenocarcinoma Hela cells. APC mRNA expression was detected by RT-PCR and Western blotting Changes of Protein and β-catenin Protein Expression in Intracellular and Nuclear. Results DAC had a concentration-dependent and time-dependent effect on the proliferation of cervical adenocarcinoma Hela cells. The IC50 of Hela cells was 28.23, 7.65 and 5.64 μmol / L at 24, 36 and 48 h respectively. The apoptotic rate of DAC-treated cervical adenocarcinoma Hela cells was (73.82 ± 0.11)%, which was significantly higher than that of the control group (12.41 ± 0.24)%, P <0.001. After treatment with HeLa cells, the proportion of S phase cells (47.82 ± 2.57)% and G2 phase cells (30.87 ± 2.28)% was significantly higher than that of blank control cells (24.08 ± 0.71)% and G2 phase cells 2.52 ± 0.84)%, P <0.001. After treatment with DAC, the demethylation status of APC gene promoter region was significantly increased and the expression of APC mRNA increased. The difference was statistically significant before and after treatment (P <0.05). After treatment with DAC, the intracellular APC protein expression was up-regulated, while the intracellular and nuclear β-catenin protein expression was down-regulated, with statistical significance (P <0.05). Conclusion DAC can induce apoptosis of cervical cancer cells by demethylation of APC gene, upregulation of intracellular APC protein expression, downregulation of intracellular and nuclear β-catenin expression.