地西他滨诱导宫颈癌细胞凋亡分子机制探讨  

Decitabine promotes the apoptosis of cervical cancer cells by induce the demethylation of APC gene

作　　者：宋汶珂[1] 唐彩霞[1] 黄彤辉[1] 孙晓红[1] 

机构地区：[1]南华大学附属南华医院妇产科,湖南衡阳421001

出　　处：《中华肿瘤防治杂志》2017年第8期540-545,共6页Chinese Journal of Cancer Prevention and Treatment

摘　　要：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。OBJECTIVE Decitabine（DAC）has good anti-tumor activity and its molecule mechanism related to DNA demethylation.This study aimed to explore the effect of apoptosis and APC gene demethylation induced by DAC and to investigate the molecular mechanism of apoptosis induced by DAC in cervical adenocarcinoma Hela cell line.METHODS DAC with different concentrations were used in cervical carcinoma Hela cells for 24,36 and 48h.MTTs were performed to detect the proliferation inhibition effects in each group to screen the concentration dependent effect and time dependent effect of proliferation inhibition for DAC in Hela cell.Flow cytometry was used to detect the effect of apoptosis and cell cycle induced by DAC in cervical carcinoma Hela cell.Before and after the treatment of DAC in cervical carcinoma Hela cells,the Methylation status of APC gene were detected by Methylation specific PCR（MSP）.mRNA expressions were detected by RT-PCR.The expression changes of APC andβ-catenin in Hela＇s intracellular and nuclear were detected by western blot.RESULTS The proliferation inhibition of DAC in cervical adenocarcinoma Hela cells had concentration dependent effect and time dependent effect.The half maximal inhibitory concentration（IC50）of Hela cells in 24,36 and 48hwere 28.23,7.65 and 5.64μmol/L,respectively.The apoptosis rate of Hela cells treated with DAC were（73.82±0.11）%,which was significantly higher than that of control group（12.41±0.24）%（P〈0.001）.After treatment of DAC,the cell ratio of Hela cells in S phase were（47.82±2.57）% and（30.87±2.28）%in G2 phase,that were significantly higher than the cell ratio of the control group in S phase（24.08±0.71）% and G2 phase（2.52±0.84）%（P〈0.001）.DAC could retard the cervical carcinoma Hela cells at the cell stage of S and G2.After the treatment with DAC,the demethylation of promoter region of APC gene increased severely.And the expression of APC mRNA was increased and the difference had statistical significance（P〈0.05）.After the

关 键 词：地西他滨 HELA细胞 甲基化 凋亡 宫颈癌 

分 类 号：R737.33[医药卫生—肿瘤]