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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 the methylation status of DAPK gene promoter and biological function in human bladder urothelial carcinoma (BUCC). Methods: BUCC 5637 cell line provided by the Chinese Academy of Sciences Cell Bank was used as a control group, and BUCC 5637 cell line treated with 10 μmol / L 5-Aza-CdR was used as the observation group. Western blot and RT-PCR were used to detect the expression of DAPK and DAPK mRNA in control group and observation group respectively. The methylation status of DAPK promoter CpG island in control group and observation group was detected by MS-PCR. Flow cytometry The cell apoptosis rate in the control group and the observation group was detected. The cell invasion ability of the control group and the observation group was detected by tumor cell invasion assay. Results: (1) The expression of DAPK in the control group was 146.32 ± 21.24, which was significantly lower than that in the observation group (312.15 ± 14.57, t = 3.582, P = 0.024); (2) DAPK mRNA expression was 0.38 ± 0.07, which was significantly lower than that of the observation group (0.82 ± 0.04, t = 4.257, P = 0.032). (3) The methylation status of DAPK promoter CpG island in the control group (4) The apoptosis rate of the control group was 2.09%, which was significantly lower than that of the observation group (6.02%), the difference was statistically significant (X2 = 0.368, P = 0.036). (5) The invasion ability of the control group was 6.12 ± 0.32, which was significantly better than that of the observation group (2.34 ± 0.12, t = 3.245, P = 0.022). Conclusion: 5-Aza-CdR can significantly increase the expression of DAPK in 5637 cells, while DAPK can significantly promote the apoptosis of BUCC cells and significantly inhibit the invasion (migration) ability of BUCC cells, suggesting that 5-Aza-CdR can be used as BUCC Gene-targeted therapies.