Wang Yan, Wang Zunsong, Li Hong, Xu Wenwei, Dong Lin, Guo Yan, Feng Saran, Bi Kehong, Zhu Chuansheng
Department of Hematology, Qianfoshan Hospital Affiliated to Shandong University, Jinan, Shandong 250014, P.R. China.
Department of Nephrology, Qianfoshan Hospital Affiliated to Shandong University, Jinan, Shandong 250014, P.R. China.
Exp Ther Med. 2017 May;13(5):2050-2055. doi: 10.3892/etm.2017.4184. Epub 2017 Mar 6.
The aim of the present study was to investigate the demethylation effect of arsenic trioxide (AsO) on the secreted frizzled-related protein 1 (SFRP1) gene and its ability to inhibit the Wingless-type MMTV integration site family (WNT) pathway in Jurkat cells. Methylation-specific polymerase chain reaction was used to examine the CpG island methylation status of the SFRP1 gene in leukemia cell lines. In addition, the effects on Jurkat cells of treatment with different concentrations of AsO for 48 h were investigated. Reverse transcription-quantitative polymerase chain reaction was employed to measure the expression of mRNAs, while western blot analysis was used to examine protein expression in cells. The SFRP1 gene was methylated in Jurkat cells. However, both methylated and unmethylated SFRP1 genes were detected in HL60 and K562 cells. In normal bone marrow mononuclear cells, the SFRP1 gene was unmethylated. Following treatment with AsO for 48 h, the SFRP1 gene was demethylated, and the mRNA and protein expression levels of the SFRP1 gene were increased. By contrast, the mRNA and protein expression levels of β-catenin and cyclin Dl were downregulated. The protein expression of c-myc was also downregulated, but AsO exhibited no significant effect on the mRNA expression of c-myc. Abnormal methylation of the SFRP1 gene was detected in Jurkat cells. These results suggest that AsO activates SFRP1 gene expression at the mRNA and protein levels in Jurkat cells by demethylation of the SFRP1 gene. Furthermore, they indicate that AsO regulates WNT target genes and controls the growth of Jurkat cells through the WNT/β-catenin signaling pathway.
本研究旨在探讨三氧化二砷（AsO）对分泌型卷曲相关蛋白1（SFRP1）基因的去甲基化作用及其抑制Jurkat细胞中无翅型MMTV整合位点家族（WNT）信号通路的能力。采用甲基化特异性聚合酶链反应检测白血病细胞系中SFRP1基因的CpG岛甲基化状态。此外，研究了不同浓度的AsO处理Jurkat细胞48小时的效果。采用逆转录定量聚合酶链反应检测mRNA表达，同时用蛋白质印迹分析检测细胞中的蛋白质表达。Jurkat细胞中SFRP1基因发生甲基化。然而，在HL60和K562细胞中检测到甲基化和未甲基化的SFRP1基因。在正常骨髓单个核细胞中，SFRP1基因未发生甲基化。用AsO处理48小时后，SFRP1基因去甲基化，SFRP1基因的mRNA和蛋白质表达水平升高。相比之下，β-连环蛋白和细胞周期蛋白D1的mRNA和蛋白质表达水平下调。c-myc的蛋白质表达也下调，但AsO对c-myc的mRNA表达无显著影响。Jurkat细胞中检测到SFRP1基因异常甲基化。这些结果表明，AsO通过使SFRP1基因去甲基化，在mRNA和蛋白质水平激活Jurkat细胞中SFRP1基因的表达。此外，这些结果表明AsO通过WNT/β-连环蛋白信号通路调节WNT靶基因并控制Jurkat细胞的生长。
