肼苯哒嗪对宫颈癌细胞系APC基因去甲基化的转录调节作用

The Study of Hydralazine on Transcription Regulation of APC Gene Demethylation in Human Cervical Cancer Cell Lines

目的：观察宫颈癌细胞系HeLa、CaSki和SiHa中APC基因的表达与甲基化修饰之间的相关性,探讨肼苯哒嗪对APC基因去甲基化的转录调节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PCR（MSP）法分析细胞中APC的甲基化状态;采用RT-PCR及FQ-PCR检测肼苯哒嗪对APC mRNA表达的影响;MTT法及流式细胞术观察肼苯哒嗪对细胞增殖及凋亡的影响;采用免疫组化SP法检测肼苯哒嗪对APC相关蛋白β-连环蛋白表达的影响。结果：APC基因在HeLa、CaSki细胞中有甲基化修饰;肼苯哒嗪能使其甲基化的APC基因重新表达;β-连环蛋白也可重新表达于细胞膜;肼苯哒嗪能抑制细胞生长,使细胞凋亡增多。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） in HeLa, CaSki and 5iHa 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： APC methylation status was analyzed using methylated specific PCR method. The expression of APC mRNA was analyzed by RT - PCR and FQ - PCR methods. MTT and FCM assays were used to observe the changes of proliferation activity and apoptosis of the cells after Hydralazine treatment. The expression of β - catenin protein which correlates closely with APC by SP method after the treatment with Hydralazine. Results： APC gene was methylated and did not express in HeLa cell, at the same time APC gene was hemimethylated and expressed less in CaSki cell. Its expression could be restored by Hydralazine. β - catenin protein can be expressed in cell membrane after the treatment with Hydralazine. Hydralazine could weaken proliferation and strengthen apoptosis of the cells. Conclusions： APC 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 ceils.