人膀胱尿路上皮癌细胞DAPK基因启动子甲基化状态与生物学功能之间的关系分析  

Analysis of relationship between promoter methylation status of DAPK gene and biological functions in bladder urothelial cell carcinoma

作　　者：宋伟[1] 周强[1] 杨金瑞[2] SONG Wei ZHOUQiang YANG Jinrui(Department of Urology, Hunan Provincial People＇s Hospital, Changsha, 410005, China Department of Urology, Second Xiangya Hospital of Central South University)

机构地区：[1]湖南省人民医院泌尿外科,长沙410005 [2]中南大学湘雅二医院泌尿外科

出　　处：《临床泌尿外科杂志》2016年第11期1016-1019,1023,共5页Journal of Clinical Urology

基　　金：湖南省科技厅科研计划项目(编号2014fj3117)

摘　　要：目的:分析人膀胱尿路上皮癌(BUCC)细胞DAPK基因启动子甲基化状态与生物学功能之间的关系。方法:以中国科学院细胞库提供的BUCC 5637细胞株作为对照组,以经10μmol/L 5-Aza-CdR处理过的BUCC 5637细胞株作为观察组。应用Western blot、RT-PCR分别检测对照组细胞及观察组细胞中DAPK、DAPKmRNA的表达;应用MS-PCR检测对照组和观察组中DAPK基因启动子CpG岛的甲基化状态;应用流式细胞术检测对照组及观察组的细胞凋亡率;应用肿瘤细胞侵袭实验检测对照组及观察组的细胞侵袭能力。结果:(1)对照组细胞中DAPK的表达为146.32±21.24,显著低于观察组的312.15±14.57,差异有统计学意义(t=3.582,P=0.024);(2)对照组细胞中的DAPKmRNA表达水平为0.38±0.07,显著低于观察组的0.82±0.04,差异有统计学意义(t=4.257,P=0.032);(3)对照组细胞中DAPK启动子基因CpG岛甲基化状态为阳性,而观察组细胞中DAPK启动子CpG岛甲基化状态为阴性;(4)对照组的细胞凋亡率为2.09%,显著低于观察组的凋亡率6.02%,差异有统计学意义(X2=0.368,P=0.036);(5)对照组细胞的侵袭能力为6.12±0.32,明显优于观察组的2.34±0.12,差异有统计学意义(t=3.245,P=0.022)。结论:5-Aza-CdR能显著增加5637细胞中DAPK的表达,而DAPK能明显促进BUCC细胞的凋亡,且显著抑制BUCC细胞的侵袭(迁移)能力,提示5-Aza-CdR可作为BUCC的基因靶向治疗药物。Objective：To analyze the relationship between promoter methylation status of DAPK gene and biological functions in human bladder urothelial cell carcinoma（BUCC）.Method：BUCC line 5637（supplied by Chinese Academy of Sciences）was divided into control group and observation group（after 10μmol/L 5-Aza-CdR treated）.Western blot,RT-PCR,and MS-PCR were performed to detect the expression of DAPK,DAPK mRNA and methylation status of DAPK in BUCC cell line 5637（control group）and urothelial cell line 5637 with 5-AzaCdR（observation group）respectively.Flow cytometry and tumour invasive assay were used to detect apoptosis and migration activity both in urothelial cell line 5637（control group）and urothelial cell line 5637 with 5-Aza-CdR（observation group）.Result：The expression of DAPK in control group was（146.32±21.24）,which was extremely lower than that in the observation group（312.15±14.57）.The difference was statistically significant（t=3.582,P=0.024）.The expression level of DAPK mRNA in control group was（0.38±0.07）,which was apparently lower than that in observation group（0.82±0.04）.The difference was statistically significant（t=4.257,P=0.032）.The methylation status of DAPK gene promoter CpG island in control group was positive,while negative in observation group.The apoptosis rate of control group（2.09%）was obviously lower than that of observation group（6.02%）,and the difference was statistically significant（χ2=0.368,P=0.036）.The invasive ability of control group（6.12±0.32）was evidently higher than that of observation group（2.34±0.12）,which the difference was statistically significant（t=3.245,P=0.022）.Conclusions：It ia suggested that 5-AzaCdR can be used as gene-targeted drug for it can significantly up-regulate DAPK expression by reversing its promoter methylation in 5637 cells.DAPK can both promote the apoptosis and inhibit the migration ability in BUCC cells.

关 键 词：膀胱尿路上皮癌 基因治疗 5-氮杂-2'-脱氧胞苷 

分 类 号：R737.14[医药卫生—肿瘤]