肼苯哒嗪对宫颈癌细胞株APC基因表达和甲基化状况以及细胞增殖与凋亡的影响

Effect of Hydralazine on demethylation status and the expression of APC gene,proliferation and apoptosis of human cervical cancer cell Iines

目的观察宫颈癌细胞株 HeLa、CaSki 和 SiHa 中 APC 基因的表达及其与甲基化修饰之间的相关性,探讨肼苯哒嗪对 APC 基因的去甲基化作用及其对细胞生长和凋亡的影响。方法应用甲基化特异性聚合酶链反应(MSP)及逆转录聚合酶链反应(RT-PCR)、荧光定量聚合酶链反应(FQ-PCR)分析肼苯哒嗪处理前后宫颈癌细胞株 APC 基因的甲基化状态及表达情况;采用免疫组织化学 SP 法检测肼苯哒嗪对宫颈癌细胞株 APC 相关蛋白β-连环蛋白表达的影响;四甲基偶氮唑蓝(MTT)法及流式细胞术观察肼苯哒嗪对宫颈癌细胞增殖及凋亡的影响。结果 (1)HeLa 及 CaSki细胞 APC 基因分别为甲基化及半甲基化,SiHa 细胞 APC 基因无甲基化。(2)40μmol/L 肼苯哒嗪处理72 h 后,HeLa、CaSki 和 SiHa 细胞的生长抑制率分别为(52.12±3.78)%、(44.31±2.59)%、(47.73±4.73)%,正常细胞人脐静脉内皮细胞(HECV)表现为耐药;HeLa 和 CaSki 细胞中 APC 基因呈去甲基化状态且 APC mRNA 均呈阳性表达;HeLa、CaSki 和 SiHa 三种细胞中 APC mRNA 表达分别比处理前增加10.35、11.40及0.73倍。(3)40 μmol/L 肼苯哒嗪作用 HeLa、CaSki 细胞72 h 后,可将细胞阻滞于 S 期和 G_2-M 期并诱导细胞凋亡;β-连环蛋白在细胞膜上有明显的表达。结论 APC 甲基化修饰是宫颈癌发生重要机制之一,肼苯哒嗪能使甲基化的 APC 基因去除甲基化修饰重新表达,且能抑制宫颈癌细胞生长。

Objective To investigate the correlation between methylation status and gene expression of APC （adenomatous polyposis coli） gene in HeLa, CaSki and SiHa cell lines of cervical carcinoma, and explore the effect of hydralazine on the transcription regulation of the 5＇CpG island demethylation of APC gene and the proliferation and apoptosis of the cell lines. Methods Methylation status and the expression of APC gene were analyzed using methylated specific PCR, RT-PCR and FQ-PCR methods. The expression of β-catenin protein which correlates closely with APC was detected by SP method after treatment with Hydralazine. MTF and FCM assays were used to observe the changes of proliferation activity and apoptosis of the cells after Hydralazine treatment. Results （ 1 ） APC gene was methylated or hemimethylated respectively in HeLa and CaSki cell lines, at the same time,APC gene was not methylated in SiHa cell. （2） After having been treated by 40 μmol/L Hydralazine for 72 hours, growth inhibitory ratios of HeLa, CaSki and SiHa cell lines were （52. 12 ＋3.78） % , （44.31 ＋2.59） % and （47.73 ＋4.73） % respectively, on the contrary, normal cell HECV＇s growth inhibitory ratio was only （27.18 ＋0.79）%. APC gene in HeLa and CaSki cell lines which were treated by 40 Ixmol/L Hydralazine for 72 hours was demethylated and expressed positively, the expression of APC mRNA in HeLa, CaSki and SiHa cell lines increased to 10. 35, 11.40 and 0.73 times respectively. （3） Hydralazine, when used at the concentration of 40 μmol/L for 72 hours, indused S phase and G2/M phase arrest and apoptosis in HeLa and CaSki cells. β-catenin protein can be expressed in cell membrane after treatment with Hydralazine. Conclusion APC gene methylation plays an important role in the carcinogenesis of cervical cells and can re-express after the treatment with Hydralazine which also could inhibit the growth of the cervical cancer cells.