抑癌基因RUNX3在膀胱癌T24细胞株中的表达以及对凋亡的影响  

Expression of Runx3 gene and its relationship with apoptosis in Human Bladder Cancer T24 Cell Line

作　　者：温机灵[1] 周祥福[2] 温机智[3] 周琦[2] 王林[2] 

机构地区：[1]上海市即墨路150号同济大学附属东方医院泌尿外科,上海200120 [2]广州市天河路600号中山大学附属第三医院泌尿外科,广州510630 [3]广州市天河路600号中山大学附属第三医院中心实验室,广州510630

出　　处：《中华腔镜泌尿外科杂志（电子版）》2008年第4期51-55,共5页Chinese Journal of Endourology(Electronic Edition)

基　　金：广东省科技计划项目(2002C30303);广州市科技局项目(2002Z3-E0017)

摘　　要：目的明确RUNX3抑癌基因在正常膀胱组织和膀胱癌T24细胞株的甲基化状态,并将野生型RUNX3基因转染T24细胞,探讨RUNX3基因恢复表达后对膀胱癌细胞凋亡的影响。方法应用RT-PCR、甲基化特异性PCR和非甲基化PCR检测正常膀胱组织、膀胱癌T24细胞中RUNX3基因的表达以及基因启动子区域CpG岛的甲基化状态。构建真核载体的RUNX3-EGFP-pDest质粒,在LipefectamineTM 2000介导下转染膀胱癌T24细胞,转染实验设立3组:空白对照组、空质粒EGFP-pDest转染组以及RUNX3-EGFP-pDest转染组。Western blot检测RUNX3蛋白表达,流式细胞仪检测细胞凋亡情况。结果正常膀胱组织RT-PCR可检测出1248bp的RUNX3基因条带,而膀胱癌T24细胞中无法检测出RUNX3基因的表达。正常膀胱组织RUNX3基因甲基化PCR检测阴性,非甲基化PCR阳性;T24细胞反之。正常膀胱粘膜组和RUNX3-EGFP-pDest质粒转染组Western blot检测,均表达RUNX3蛋白,而膀胱癌T24细胞未表达RUNX3蛋白。流式细胞仪检测,空白对照组的凋亡率为(3.1±0.46)%,而EGFP-pDest转染组和RUNX3-EGFP-pDest质粒转染组的凋亡率分别为(10.1±1.62)%、(41.20±1.53)%,应用方差分析的LSD法和SNK法进行均数的多重两两比较,RUNX3-EGFP-pDest质粒转染组与EGFP-pDest转染组、空白对照组凋亡率之间的差异均具有显著性,P<0.01。结论正常膀胱组织RUNX3基因正常表达,未发生启动子区域CpG岛甲基化,而膀胱癌T24细胞可能因RUNX3基因启动子区域CpG岛发生甲基化,致RUNX3基因无法表达。转染野生型RUNX3抑癌基因后促进了膀胱癌T24细胞的凋亡。Objective To identify the methylation status of runt-related transcription factor 3 （RUNX3） anti-oncogene in normal bladder tissue and T24 bladder tumor cell line, and discuss the effects of re-expression of RUNX3 gene on bladder tumor cell apoptosis. Methods We transfected wild-type RUNX3 gene to T24 bladder tumor cell line and detected apoptosis by flow cytometry.The status of RUNX3 gene expession and methylation of CpG islands in the promoter regions was examined by RT- PCR ,methylation-specific PCR and unmethylation PCR respectively. The constructed eukaryotic RUNX3-EGFP-pDest vector was transfected into T24 bladder tumor cell line via lipofectamine.There were 3 groups：null control group （no transfection）;empty plasmid （EGFP-pDest） transfection groupand and eukaryotic vector plasmid （RUNX3-EGFP-pDest） transfecdon group.The expression of RUNX3 gene was assessed by Western-blotting.Cell apoptosis was analyzed by flow cytometry. Results Assessment of RUNX3 expression in normal bladder tissue and bladder tumor cell line revealed a distinct pattern of expression. Analyzed by the flow cytometry ,the apoptotic ratio of the RUNX3-EGFP-pDest group （41.20±1.53）% was significantly higher than the empty plasmid transfection group （10.1±1.62）% and null control group（3.1±0.46）%（P〈0.01）. Conclusion RUNX3 gene expresses normally in healthy bladder tissue without CpG islands methyladon in promoter regions ,whereas it can not express in T24 bladder tumor cell line due to methylation of CpG islands in the promoter regions . Transfection of wild-type RUNX3 gene can induce the apoptosis of T24 bladder tumor cell line.

关 键 词：膀胱肿瘤 抑癌基因 T24细胞 甲基化 

分 类 号：R737.14[医药卫生—肿瘤]