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目的分析DAPK抑癌基因在T24、5637、ScaBER三种膀胱癌细胞中的表达情况及该基因启动子区的甲基化状态。方法采用半定量RT-PCR和Western blot分别检测三种膀胱癌细胞中DAPK基因的表达,用甲基化特异PCR(MSP)方法检测三种细胞中的DAPK基因启动子甲基化状态。观察甲基转移酶(DMNT)抑制剂5-aza-2’-deoxy-eytidine(5-aza-CdR)对以细胞中的基因表达和甲基化状态的影响。结果T24细胞中未检测到DAPK基因的表达,也未检测到启动子甲基化;5637细胞中该基因表达水平极低,在SeaBER细胞中该基因的表达较前两者要高。5637细胞和ScaBER细胞中均有甲基化改变。2.5μmol/l浓度的5-aza-CdR可以有效上调5637和SeaBER细胞的DAPK基因的表达。结论抑癌基因DAPK的表达异常与移行细胞癌的发生发展有重要联系,且基因启动子区CpG岛的异常甲基化在调节DAPK基因的表达中发挥重要作用。
Objective To analyze the expression of DAPK tumor suppressor gene in three T24, 5637 and ScaBER bladder cancer cells and the methylation status of the promoter region of DAPK. Methods DAPK gene expression in three kinds of bladder cancer cells was detected by semi-quantitative RT-PCR and Western blot. Methylation-specific PCR (MSP) was used to detect the methylation status of DAPK promoter in the three cell lines. The effect of Methyltransferase (DMNT) inhibitor 5-aza-2’-deoxy-eytidine (5-aza-CdR) on gene expression and methylation status in cells was observed. Results No DAPK gene expression or promoter methylation was detected in T24 cells. The expression level of this gene was extremely low in 5637 cells, which was higher in SeaBER cells. Metabolic changes were found in both 5637 cells and ScaBER cells. The concentration of 2.5-μmol / l 5-aza-CdR effectively up-regulated the DAPK gene expression in 5637 and SeaBER cells. Conclusion The abnormal expression of DAPK gene is closely related to the occurrence and development of transitional cell carcinoma. Aberrant methylation of CpG island in gene promoter plays an important role in the regulation of DAPK gene expression.