si-DNMT1 restore tumor suppressor genes expression through the reversal of DNA hypermethylation in cholangiocarcinoma
si-DNMT1 restore tumor suppressor genes expression through the reversal of DNA hypermethylation in cholangiocarcinoma
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DOI：
10.1016/j.clinre.2013.11.004
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发表时间：
2014-04-01
影响因子：
2.7
通讯作者：
Wang, Jiming
中科院分区：
文献类型：
--
作者：
Xiang, Jifeng;Luo, Fang;Wang, Jiming
目的：本研究旨在探讨shorthairpin RNA质粒载体敲低人DNA甲基转移酶1基因对肝门部胆管癌细胞增殖及抑癌基因甲基化状态和表达的影响。用靶向人DNA甲基转移酶1的shorthairpin RNA质粒载体转染QBC 939细胞。在不同时间点收集对照和人DNA甲基转移酶1 shorthairpin RNA质粒载体转染的细胞，逆转录-聚合酶链反应检测人DNA甲基转移酶1和肿瘤抑制基因（细胞周期蛋白依赖性激酶抑制因子2B、细胞周期蛋白依赖性激酶抑制因子2A、RAS相关结构域家族1和钙粘蛋白-1）的表达水平。此外，通过Western印迹证实了干扰效率。采用甲基化特异性聚合酶链反应检测抑癌基因甲基化状态。此外，人DNA甲基转移酶1敲低对增殖的影响通过3-（4，5-二甲基噻唑-2-基）-2，5-二苯基溴化四唑鎓含量测定。人DNA甲基转移酶1的靶向基因敲除恢复了肿瘤抑制基因细胞周期蛋白依赖性激酶抑制剂2B、细胞周期蛋白依赖性激酶抑制剂2A、RAS相关结构域家族1和钙粘蛋白-1的表达水平，这表明这些肿瘤抑制基因的沉默与启动子高甲基化有关。结论：靶向敲低人DNA甲基转移酶1表达可恢复肿瘤抑制基因的表达水平，从而抑制QBC 939细胞的增殖。这些结果可能为胆管癌新疗法的开发提供见解。(C)2013年Elsevier Masson SAS。All rights reserved.
Objective: The aim of our study was to evaluate the effect of shorthairpin RNA plasmid vector knockdown of human DNA methyltransferase 1 on proliferation and the methylation status and expression of tumor suppressor genes in hilar cholangiocarcinoma.Methods: The hilar cholangiocarcinoma cell line QBC939 was utilized for this study. QBC939 cells were transfected with a shorthairpin RNA plasmid vector targeting human DNA methyltransferase 1. Control and human DNA methyltransferase 1 shorthairpin RNA plasmid vector-transfected cells were collected at different time points, and the expression levels of human DNA methyltransferase 1 and tumor suppressor genes (cyclin-dependent kinase inhibitor 2B, cyclin-dependent kinase inhibitor 2A, RAS association domain family 1, and cadherin-1) were detected by reverse transcription-polymerase chain reaction. Furthermore, interfering efficiency was confirmed by Western blotting. The methylation status of tumor suppressor genes was detected using methylation-specific polymerase chain reaction. Furthermore, the effect of human DNA methyltransferase 1 knockdown on proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Results: Targeted gene knockout of human DNA methyltransferase 1 restored the expression levels of tumor suppressor genes cyclin-dependent kinase inhibitor 2B, cyclin-dependent kinase inhibitor 2A, RAS association domain family 1, and cadherin-1, indicating that the silencing of these tumor suppressor genes is associated with promoter hypermethylation. In addition, knockdown of human DNA methyltransferase 1 expression significantly inhibited the proliferation of QBC939 cells.Conclusions: Targeted knockdown of human DNA methyltransferase 1 expression restores the expression levels of tumor suppressor genes, thus inhibiting the proliferation of QBC939 cells. These results may provide insight for the development of novel therapies for cholangiocarcinoma. (C) 2013 Elsevier Masson SAS. All rights reserved.