肼曲嗪和丙戊酸对胆管癌ASC/TMS1基因甲基化的影响

The effects of hydralazine and valproic acid on the methylation of ASC/TMS1 of human cholangiocarcinoma cell line

目的研究甲基化酶抑制剂肼曲嗪和组蛋白脱乙酰化酶抑制剂丙戊酸联合干预对胆管癌细胞QBC939,ASC/TMS1基因甲基化调控的影响,并探讨caspase-1介导的细胞凋亡与ASC/TMS1甲基化的关系。方法利用肼曲嗪和丙戊酸单独或联合干预胆管癌细胞QBC939,用Annexin-FITC和Pro-pidium双染检测干预后细胞凋亡率;用RT-PCR和MSP技术检测干预后ASC/TMS1基因甲基化状态的改变和mRNA的转录水平,用RT-PCR检测干预后caspase-1的mRNA转录水平。结果单用肼曲嗪或丙戊酸盐干预对ASC/TMS1表达无明显恢复,而联用以上两药后ASC/TMS1表达明显增加(P<0.05)。两药合用48h组基因表达量高于合用24h组表达量(P<0.05);且caspase-1表达也明显增加(P<0.05),胆管癌细胞生长明显受抑制,凋亡率明显增加(49.88±0.044)%。结论肼曲嗪和丙戊酸联合干预对ASC/TMS1去甲基化有明显的协同作用。两药联用后胆管癌细胞凋亡率的增加可能系因去甲基化后ASC/TMS1基因表达增加,通过caspase-1途径诱导细胞凋亡。

Objective To investigate changes of methylation status of ASC/TMS1 in QBC939 cell line of cholangiocarcinoma before and after combined DNA methylation and histone deacetylase inhibitors treatment, and the correlation of the apoptosis which is induced by caspase-1 , and methylation status of ASC/TMS1. Methods Apoptosis was detected by mixed dye including both Annexin-FITC and Propidium with flow cytometry technique, and changes of methylation and transcription of mRNA were explored by RT-PCR and MSP techniques after the intervention of hydralazine and valproic acid either alone or combined for 24 hours and 48hours, Results The transcription of mRNA of TMS1/ASC gene and caspase-1 re-expressed again after the combined intervention of hydralazine and valproic acid, which was higher than that of the cells treated with either hydralazine or valproate alone （ P 〈 0. 05 ） . The demethylation effect of 48 h by combind intervention treatment was better than that of 24h （ P 〈 0, 05 ）, The growth of the QBC939 cell line was inhibited, and flow cytometry showed marked increase of apoptosis （ 49.88 ± 0. 044 ） %, Conclusions TMS 1 / ASC gene and caspase-1 may re-express after the synergistical intervention of hydralazine and valproic acid, and the effect is more obvious as the treatment time is extended. The apoptosis of QBC939 cell line is increased, which may be indued by caspase-1 passway.