透明细胞肾细胞癌组织中DNMT3B异构体表达及其整体甲基化的分析

Expression of DNA methyltransferase 3B isoforms and global methylation level in clear cell renal cell carcinoma

目的检测透明细胞肾细胞癌组织中DNA甲基转移酶3B(DNA methyltransferase 3B,DNMT3B)异构体表达及其整体甲基化的变化,探讨这些变化与透明细胞肾细胞癌的关系。方法选取15例透明细胞肾细胞癌及其癌旁组织,应用Real-time PCR技术检测DNMT1、DNMT3A、DNMT3B与DNMT3B六种主要异构体的mRNA表达,应用Western blot法检验DNMT3B4蛋白表达;应用联合亚硫酸氢钠的限制性内切酶分析法(COBRA)分析重复序列Alu和LINE-1的甲基化水平。结果透明细胞肾细胞癌组织中DNMT3B4 mRNA和蛋白表达水平比癌旁组织高;重复序列Alu和LINE-1的甲基化水平在透明细胞肾细胞癌组织中比癌旁组织低。结论 DNMT3B4表达增加可能与整体甲基化降低及透明细胞肾细胞癌的发生有密切关系。

Purpose The aim of this study was to detect the expressions of DNA methyltransferase 3B isoforms and global methylation level in clear cell renal cell carcinoma （ccRCC） and to discuss the relationship between the changes of them and the ccRCC. Methods Fifteen pairs of tissue samples, which included ccRCC tissue and adjacent matched normal tissue from each patient, were used in this study. Real-time PCR was used to measure the mRNA expression levels of DNMT1, DNMT3A, total DNMT3B, and six kinds of DNMT3B variants. Western blot was used to detect the protein level of DNMT3B4. The repetitive sequences DNA methylation level of Alu and long interspersed nuclear elements （ LINE-1 ） were measured by combine bisulfite restriction analysis （COBRA）. Results Both mRNA and protein levels of DNMT3B4 were increased in ccRCC tissue compared with control tissue. Additionally, Alu elements and LINE-1 were hypomethylated in renal cell carcinoma tissue. Conclusion Overexpression of DNMT31M may play an important role in human kidney tumorigenesis through the decrease of global methylation.