肼苯哒嗪对宫颈癌细胞系侵袭力的影响

Effect of Hydrazine Demethylation on Invasion of Human Cervical Cancer Cells

目的探讨肼苯哒嗪去甲基化作用对人宫颈癌细胞系HeLa(HPV18型)、CaSki和SiHa(HPV16型)细胞侵袭力的抑制影响。方法 Transwell侵袭小室法检测肼苯哒嗪处理前后人宫颈癌细胞系HeLa、CaSki和SiHa的侵袭力。甲基化特异性PCR(MSP)法检测肼苯哒嗪处理前后各细胞系中APC基因和CDH1基因5′端启动子区CpG岛甲基化状态。实时荧光定量PCR(FQ-PCR)法检测肼苯哒嗪处理前后,上述三种细胞系中APC和CDH1mRNA表达情况。结果与未处理组比较,10μmol/L肼苯哒嗪对人宫颈癌细胞系HeLa、CaSki和SiHa的侵袭力抑制作用差异无统计学意义,20μmol/L与40μmol/L肼苯哒嗪对人宫颈癌细胞系HeLa、CaSki和SiHa的侵袭力抑制作用差异有统计学意义(P<0.05,P<0.01);且不同浓度之间有差异,以40μmol/L肼苯哒嗪抑制作用最强。40μmol/L肼苯哒嗪处理后,上述三种细胞系中APC和CDH1基因呈现不同程度的去甲基化现象。经40μmol/L肼苯哒嗪处理后,上述三种细胞系中APCmRNA表达分别是处理前的5.89倍、8.46倍及0.97倍,CHD1mRNA表达分别是处理前的4.82倍、5.90倍及8.46倍。结论一定浓度的肼苯哒嗪能明显抑制宫颈癌细胞系HeLa、CasKi和SiHa的侵袭力,其作用机制之一是通过去甲基化作用影响APC和CHD1的表达。

Objective To investigate suppression effect of hydrazine demethylation on invasive activity of human cervical cancer cell lines HeLa（HPV18 type）,CaSki and SiHa（HPV16 type）.Methods Invasive activity of HeLa,CaSki and SiHa cell lines of human cervical cancer treated with Hydralazine（Hyd） of different concentration were analyzed by using millipore cells invasion room method.The methylation status of 5＇CpG island in the promotor of APC and CDH1 genes in the three cell lines were analyzed using MSP method before and after Hyd treatment.The expression of APC and CDH1 mRNA in the three cell lines was studied by FQ-PCR methods before and after Hyd treatment.ResultsComparing with the normal control group,suppressing invasive activity of HeLa,CaSki and SiHa cell lines of human cervical cancer treated with 10 μmol/L Hyd have no statistical significance,while the group with 20 μmol/L Hyd have statistical significance（P0.05）,and the group with by 40 μmol/L Hyd have significant difference（P0.01）.Different drug density being compared,the group treated with 40 μmol/L Hyd have significance suppression.After 40 μmol/L Hyd treatment for 48h,methylation of the APC and CDH1 genes of these three cell lines was at different levels of demethylation.Expression of APC and CDH1 mRNA was up-regμlated in these three cell lines after treatment.The expression of APC mRNA in HeLa,CaSki,and SiHa cell lines increased 5.89-,8.46-,and 0.97-fold,and CDH1 mRNA increased 4.82-,5.90-,and 8.46-fold,respectively.Conclusion Hydralazine has an effect on suppressing invasive activity of HeLa,CaSki and SiHa cell lines of human cervical cancer,one of the mechanisms of action may be demethylation.