Increased CXCR4 expression in AsPC1 pancreatic carcinoma cells with RNA interference-mediated knockdown of DNMT1 and DNMT3B
Increased CXCR4 expression in AsPC1 pancreatic carcinoma cells with RNA interference-mediated knockdown of DNMT1 and DNMT3B
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DOI：
10.1016/j.biopha.2009.06.008
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发表时间：
2010-04-01
影响因子：
7.5
通讯作者：
Jagodzinski, P. P.
中科院分区：
文献类型：
--
作者：
Przybylski, M.;Kozlowska, A.;Jagodzinski, P. P.
DNA 甲基化对 CXCR4 表达的影响已在胰腺癌和黑色素瘤细胞中得到证实，但人们对 DNA 甲基转移酶 1 和 3（DNMT1 和 DNMT3B）对 CXCR4 表达的影响知之甚少。利用慢病毒载体，我们在 AsPC1 胰腺癌细胞中创建了稳定的 RNA 干扰介导的 DNMT1 和 DNMT3B 敲低。使用逆转录实时定量 PCR 和流式细胞术分析，我们评估了这些细胞中 CXCR4 转录物表达和蛋白质水平的增加。亚硫酸氢盐测序分析表明，在 DNMT1 敲低的细胞中，启动子去甲基化水平似乎比 DNMT3B 敲低的细胞更有效。此外，DNMT1 和 DNMT3B 的联合 RNA 干扰敲低增加了启动子去甲基化，导致 CXCR4 表达略有增加。然而，去甲基化剂5-Aza-2'-脱氧胞苷对启动子去甲基化表现出最强的作用，这与AsPC1细胞中CXCR4转录物和蛋白质的最高产量相关。我们的结果表明，DNMT1 在维持 CXCR4 启动子甲基化中起主要作用，而 DNMT3B 可能作为辅助 DNA 甲基转移酶来调节 AsPC1 细胞中 CXCR4 的表达。 (C) 2009 Elsevier Masson SAS。版权所有。
The effect of DNA methylation on CXCR4 expression has been demonstrated in pancreatic cancer and melanoma cells, but little is known about the effect of DNA methyltransferases 1 and 3 (DNMT1 and DNMT3B) on CXCR4 expression. Employing lentiviral vectors, we created stable RNA interference-mediated knockdown of DNMT1 and DNMT3B in AsPC1 pancreatic cancer cells. Using reverse transcription real-time quantitative PCR and flow cytometric analysis, we evaluated the increase in the expression of CXCR4 transcript and protein levels in these cells. Bisulfite sequencing analysis showed that the level of promoter demethylation appeared more effective in cells with knockdown of DNMT1 than in those with DNMT3B knockdown. Furthermore, the combined RNA interference knockdown of both DNMT1 and DNMT3B increased promoter demethylation, leading to a slight increase in CXCR4 expression. However, the demethylating agent 5-Aza-2'-deoxycytidine exhibited the strongest effect on promoter demethylation, which correlated with the highest production of CXCR4 transcript and protein in AsPC1 cells. Our results indicate that DNMT1 plays the main role in maintenance of methylation of CXCR4 promoter, while DNMT3B may function as an accessory DNA methyltransferase to modulate CXCR4 expression in AsPC1 cells. (C) 2009 Elsevier Masson SAS. All rights reserved.