氯普鲁卡因诱导体外人宫颈癌细胞抑癌基因CDH1、APC和P16启动子去甲基化及基因表达

Effects of chloroprocaine on the methylation status and the expression of CDH1, APCand P16genes in human cervical cancer cells in vitro

目的观察氯普鲁卡因(CP)对人宫颈癌细胞HeLa和CaSki的抑癌基因CDH1、APC及P16启动子甲基化水平和基因表达的影响。方法用不同终浓度的CP(0、1、1.5、2、3和4 mmol/L)处理癌细胞系HeLa和CaSki及正常人脐静脉内皮细胞HUVEC,MTT法检测细胞生长抑制率;用甲基化特异性PCR(MSP)和RT-PCR检测1.5 mmol/LCP处理后各细胞CDH1、APC及P16启动子甲基化状态及基因表达水平。结果 1.5 mmol/L CP作用96 h后,HeLa和CaSki抑制率分别为66.17%±5.82%和69.12%±6.89%,显著高于HUVEC的21.78%±3.12%,1.5 mmol/LCP处理宫颈癌细胞96 h后,CDH1、APC及P16基因启动子均有不同程度去甲基化,3种基因mRNA均增强或者恢复了表达。结论 CP可以抑制人宫颈癌细胞HeLa和CaSki增殖,诱导HeLa和CaSki的CDH1、APC及P16基因启动子去甲基化,并可增加或者恢复相应基因的表达。

Objective To investigate the effects of chloroprocaine （CP） on the methylation status and the expres- sion of CDH1, APC and P16 genes in human cervical cancer cell lines HeLa and CaSki. Methods HeLa, CaSki and the normal human cell HUVEC cultured in vitro were exposed to different concentrations （0, 1, 1.5, 2, 3 and 4 retool/L） CP. The growth inhibition of the three cell lines treated at 48, 72 and 96 h were studied by MTF as- say. The three cell lines were all treated by the certain concentration CP which inhibited the growth of HeLa and CaSki significantly but not affect the growth of HUVEC apparently. The methylation status and the expression of CDH1, APC and P16 genes in the three cell lines were analyzed by methylated specific-PCR （MSP） and RT-PCR, respectively. Results After being treated by 1.5 mmol/L CP for 96 h, the inhibition rates of HeLa and CaSki were 66. 17% ±5.82% and 69.12% ±6.89% , which were significantly higher than that of HUVEC, 21.78% ± 3.12%. CDH1, APC and P16 genes were all demethylated at different levels and mRNA expression of the three genes were recovered or increased in HeLa and CaSki cells after treated by 1.5 mmoL/L CP for 96 h. ConclusionsCP may inhibit the growth of HeLa and CaSki cells, meanwhile, it could demethylate CDH1, APC and P16 genes and recover or increase the genes＇ expression in the two cervical cancer ceils.