Objective To detect the growth differentiation 15 factor(GDF15) methylation status and explore the role of promoter region aberrant methylation in the development of bladder cancer.Methods We detected the methylation status of GDF15 promoter CpG island(CGI)in bladder cancer cell line 5637,HT1376,KU19-19,normal bladder tissue and 1 pairs of bladder tissues(cancer and para-cancerous tissue) by using bisulfite genomic sequencing PCR(BSP) combined with TA clone for sequencing.The methylation rate of GDF15 in 5637 was compared before and after treatment of the inhibitor of DNA methyltransferase(5-Aza-2-deoxycitydine,5-Aza-dc).Western blot was used to detect the protein expression of GDF15;MTT was used to detect cell proliferation;scratch assay was used to detect cell migration and transwell assay was used to detect cell invasion capability.Results The average methylation rate of GDF15 promoter CGI was 86.91% in 5637, 10.71% in HT1376, 8.33% in KU19-19, and 86.91% in cancer tissues,9.52% in para-cancerous tissues, 5.95% in normal tissues.The methylation rate of cancer tissue was higher than that of normal bladder and para-cancerous tissues(P<0.05).5-Aza-dc could reverse the methylation status of GDF15 promoter in 5637,and compared with before treatment all of the index including of protein increasing, cell proliferation, migration and invasion ability decreasing had statistical significance(P<0.05).Conclusion This promoter hypemlethylation is correlated with GDF15 gene expression in bladder cancer cell line 5637 and bladder tissues,and plays a key role in GDF15 silencing.Reverse methylation status can result in protein increasing, cell proliferation, migration and invasion ability decreasing.Aberrant hypermethylation of GDF15 might become early diagnosis index and treatment target of bladder cancer.%目的 检测生长分化因子15(GDF15)在膀胱癌中的甲基化状态,探讨其启动子区异常甲基化对于膀胱癌发生发展的作用.方法 应用重亚硫酸盐测序PCR(BSP)联合T-载体PCR产物(TA)克隆检测人膀胱移行细胞癌细胞系5637、HT1376、KU19-19、膀胱癌组织、癌旁组织、正常组织样品中GDF15启动子区甲基化状态,并用甲基化酶抑制剂5-Aza-2-deoxycitydine(5-Aza-dc)处理5637,观察处理前后平均甲基化率的变化情况,Western blot法检测5637处理前后蛋白表达情况,MTT法检测细胞增殖情况,划痕实验检测迁移情况,Transwell法检测侵袭能力.结果 5637、HT1376、KU19-19细胞中GDF15启动子区平均甲基化率为89.29%、10.71%、8.33%,肿瘤组织、癌旁组织及正常组织分别为86.91%、9.52%、5.95%,膀胱癌组织中 GDF15启动子区甲基化率较癌旁组织和正常组织中高,差异有统计学意义(P<0.05);5-Aza-dc可以逆转5637中GDF15的甲基化状态:与处理前相比,处理组5637细胞系GDF15蛋白表达增加,增殖、迁移、侵袭能力下降,差异有统计学意义(P<0.05).结论 膀胱癌中GDF15基因表达与甲基化状态有关,启动子区高甲基化导致基因沉默,逆转甲基化状态可以使基因蛋白表达增加,细胞增殖、迁移、侵袭能力均降低.GDF15基因甲基化异常状态有可能成为膀胱癌诊断的潜在靶点.
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