膀胱癌中GDF15基因启动子区甲基化检测及逆转其甲基化状态对5637膀胱癌细胞增殖、迁移、侵袭的影响

发布时间：2018-04-27 11:06

本文选题：膀胱癌 + 甲基化　；参考：《安徽医科大学学报》2017年07期

【摘要】：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(P0.05);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(P0.05)。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。
[Abstract]:Objective to detect the methylation status of growth differentiation factor 15 (GDF 15) in bladder cancer and to explore the role of abnormal methylation in its promoter region in the occurrence and development of bladder cancer. Methods the methylation status of GDF15 promoter was detected in human bladder transitional cell carcinoma cell line 5637 (HT1376) KU19-19 by PCRS-BSPand T- vector PCR product (TAA). The methylation status of GDF15 promoter was detected in bladder cancer tissues, adjacent tissues and normal tissues. 5637 was treated with 5-Aza-2-deoxycineine (5-Aza-2-deoxycineine). The changes of average methylation rate before and after treatment were observed. Western blot assay was used to detect protein expression before and after treatment. MTT assay was used to detect cell proliferation, and scratch test was used to detect migration and invasion ability. Results the average methylation rate of GDF15 promoter in 5637 HT1376 KU19-19 cells was 89.290.71 and 8.33. The methylation rate of GDF15 promoter in bladder cancer was higher than that in paracancerous and normal tissues. The difference was statistically significant (P 0.05) that 5-Aza-dc could reverse the methylation of GDF15 in 5637. Compared with before treatment, the expression of GDF15 protein increased, proliferation, migration and invasion decreased in 5637 cell line of treatment group, and the difference was statistically significant (P 0.05). Conclusion GDF15 gene expression is related to methylation status in bladder cancer. Hypermethylation in promoter region leads to gene silencing. Reverse methylation can increase gene protein expression, cell proliferation and migration. Decreased invasiveness of the. GDF15 gene methylation may be a potential target for the diagnosis of bladder cancer.
【作者单位】：安徽医科大学附属安徽省立医院泌尿外科;
【基金】：安徽省自然科学基金(编号:1608085MH166)
【分类号】：R737.14

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