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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 observe the expression of APC gene in cervical cancer cell lines HeLa, CaSki and SiHa and its relationship with methylation modification, and to explore the demethylation of APC gene by hydralazine and its effect on cell growth and apoptosis The impact of death. Methods Methylation specific polymerase chain reaction (MSP), reverse transcription polymerase chain reaction (RT-PCR) and fluorescence quantitative polymerase chain reaction (FQ-PCR) were used to analyze the effect of hydralazine treatment on cervical cancer cell line APC The methylation status and expression of APC-related protein in cervical cancer cell lines were detected by immunohistochemical SP method. The effects of hydralazine on the expression of APC-related protein β-catenin in cervical cancer cell lines were detected by MTT assay and flow cytometry Effect of Hydralazine on Proliferation and Apoptosis of Cervical Cancer Cells by. Results (1) The APC genes of HeLa and CaSki cells were methylated and methylated, respectively. There was no methylation of APC gene in SiHa cells. (2) The growth inhibition rates of HeLa, CaSki and SiHa cells were (52.12 ± 3.78)%, (44.31 ± 2.59)% and (47.73 ± 4.73)% respectively after treated with 40μmol / L hydralazine for 72 h The expression of APC mRNA in HeLa and CaSki cells was demethylated and APC mRNA was positive. The expression of APC mRNA in HeLa, CaSki and SiHa cells was lower than that before treatment Increase 10.35,11.40 and 0.73 times. (3) After treated with 40 μmol / L hydralazine for 72 h, HeLa and CaSki cells could arrest cells in S phase and G 2 -M phase and induce apoptosis; β-catenin was significantly expressed on the cell membrane . Conclusion APC methylation modification is one of the important mechanisms of cervical cancer. Hydralazine can re-express the methylated APC gene and inhibit the growth of cervical cancer cells.