甲基化修饰对人宫颈癌细胞系中fhit基因的调控.docVIP

甲基化修饰对人宫颈癌细胞系中FHIT基因的调控 【摘要】 目的探讨FHIT在子宫颈癌发生中的作用及FHIT基因失活的机制。方法培养宫颈癌细胞C33A、HeLa、CasKi、SiHa 及人脐静脉血管内皮细胞ECV304，进行5azadC干预前后FHIT基因在5株细胞中的甲基化分析，并通过逆转录聚合酶链式反应和免疫荧光化学染色方法检测5azadC干预前后FHIT基因在5株细胞中的表达。结果4种宫颈癌细胞均存在FHIT基因的甲基化，正常对照细胞ECV304在5azadC干预前后均未见明显FHIT基因扩增产物。4株宫颈癌细胞在5azadC化学干预前均未见明显FHIT的荧光着色，而干预后的细胞胞浆中可见FHIT的表达强度明显增强，尤其在106M及2×106M干预浓度下的表达最强(Plt;0.05)；干预48 h、72 h后FHIT 的表达与干预24 h的类似。ECV304细胞在5azadC化学干预前后的胞浆均可见到FHIT的阳性表达，其表达强度之间无明显差异(Pgt;0.05)。结论FHIT基因的甲基化在宫颈癌中是频发事件，甲基化可能是FHIT 基因沉默及宫颈癌发生的重要机制。 【关键词】 脆性组氨酸三联体基因（FHIT）；人宫颈癌细胞系；5azadC；DNA甲基化；免疫荧光 　　Pattern of FHIT Gene 5’ CpG Island Methylation Contribute to Human Cervical Carcinoma Cell Tumorigenesis 　　Abstract：Objective To determine whether hypermethylation of FHIT played an important role in cervical tumorigenesis.MethodsBy incubating DNA in the presence of a methylase, we investigated the methylation of FHIT in four cervical cancer cell lines and one human umbilical vein endothelial cell line treating with the DNA methyltransferase inhibitor, 5aza2deoxycytidine(5azadC). The expression of FHIT was also monitored by RTPCR and immunofluorescence technique before and after 5azadC treatment.ResultsWe found that methylation of FHIT occured in the four cervical cancer cell lines instead of the ECV304. Through RTPCR and immunofluorescence technique, we found that the expression of FHIT, which could hardly be detected in all four cervical cancer cells, was significant different after 5azadC treatment. However, the ECV304 cell expressed FHIT at constant levels before and after 5azadC treatment.ConclusionWe concluded that hypermethylation of FHIT gene occurred frequently in cervical tumorigenesis. And hypermethylation within the regulatory sequences of FHIT gene might be an important means for its inactivation in cervical cancer. 　　Key words：Fragile Histidine Triad Gene (FHIT); Human cervical cancer cell lines; 5aza2deoxycytidine; DNA methylation; Imm