地西他滨对AML1-ETO阳性急性髓系白血病细胞中PD-L2分子表达的影响

李梦月; 邵杨柳; 李雨晴; 王莉莉; 高晓宁

doi:10.3969/j.issn.2095-5227.2023.03.007

地西他滨对AML1-ETO阳性急性髓系白血病细胞中PD-L2分子表达的影响

Effect of decitabine on expression of PD-L2 gene in AML1-ETO positive acute myeloid leukemia cells

摘要

摘要:
背景复发难治性AML1-ETO融合基因阳性的急性髓系白血病(acute myeloid leukemia，AML)患者预后差。程序性细胞死亡蛋白-配体2 (programmed cell death-ligand 2，PD-L2)是参与白血病免疫逃逸的重要分子，研究去甲基化药物对白血病细胞中PD-L2分子表达的影响，对于优化用药方案具重要意义。
目的分析AML1-ETO融合基因阳性的AML细胞中PD-L2分子启动子区甲基化水平及DNA甲基转移酶抑制剂地西他滨(decitabine，DAC)对其表达的影响。
方法以AML1-ETO融合基因阳性AML细胞系Kasumi-1以及两对AML1-ETO沉默(SKNO-1-PGK，SKNO-1-siA/E)或诱导细胞系(U937-MT，U937A/E，U937A/E + ZnSO₄)为模型，采用终浓度为2.5 μmol/L的DAC或0.1%二甲基亚砜(对照)处理细胞72 h，其中Kasumi-1、SKNO-1-PGK细胞系设置0.25 μmol/L、1.0 μmol/L和2.5 μmol/L 3个浓度梯度。采用亚硫酸氢盐修饰后测序法检测PD-L2 DNA启动子区特异性位点的甲基化水平；采用逆转录实时定量PCR技术检测PD-L2 mRNA表达水平。
结果 Kasumi-1、SKNO-1-PGK细胞系中PD-L2 DNA启动子区呈高甲基化水平，DAC处理后该区域DNA甲基化水平下降(94.3% vs 90.7%，88.6% vs 83.6%)。2.5 μmol/L DAC处理后的Kasumi-1、SKNO-1-PGK、SKNO-1-siA/E、U937A/E细胞中PD-L2 mRNA的相对表达水平均显著高于对照组(P均＜0.05)；特别是DAC处理后，沉默AML1-ETO基因的细胞系中PD-L2表达增加(SKNO-1-siA/E与SKNO-1-PGK相比上升35.80%，P=0.031)，过表达AML1-ETO基因的细胞系中PD-L2表达减少(U937A/E + ZnSO₄与U937A/E相比下降95.51%，P=0.036)。经DAC处理后的Kasumi-1和SKNO-1-PGK细胞系中，PD-L2 mRNA表达呈浓度依赖性递增(Kasumi-1：r²=0.87；SKNO-1-PGK：r²=0.93。P均＜0.05)。
结论 AML1-ETO阳性AML细胞中PD-L2基因的表达受DNA甲基化调控，AML1-ETO可能参与了PD-L2基因表达的抑制。

Abstract:
Background Refractory and relapsed acute myeloid leukemia patients carrying AML1-ETO fusion gene have a poor prognosis. Programmed cell death-ligand 2 (PD-L2) is an important molecule involved in leukemia immune escape. Studying the effect of demethylation agents on immune checkpoint molecules in the leukemia cells will help to optimize the treatment regimen.
Objective To analyze the DNA methylation level of PD-L2 gene promoter in AML1-ETO-positive AML cells and investigate the effect of DNA methyltransferase inhibitor decitabine (DAC) on the expression of PD-L2.
Methods The AML1-ETO-positive AML cell lines Kasumi-1 and two pairs of AML1-ETO manipulated cell lines (AML1-ETO silenced: SKNO-1-PGK, SKNO-1-siA/E and AML1-ETO induced: U937-MT, U937A/E, U937A/E + ZnSO₄) were used as the model. The cells were treated with DAC with a final concentration of 2.5 μmol/L or 0.1% dimethyl sulfoxideas (vehicle control) for 72h. Kasumi-1 and SKNO-1-PGK cells were treated with different concentrations of DAC (0.25 μmol/L, 1.0 μmol/L and 2.5 μmol/L). DNA methylation levels of the PD-L2 gene promoter in Kasumi-1 and SKNO-1-PGK cell lines were detected by bisulfite sequencing method. Reverse transcription and quantitative PCR technology was used to detect PD-L2 mRNA levels in each cell lines.
Results The PD-L2 promoter region in Kasumi-1 and SKNO-1-PGK cell lines showed high DNA methylation levels (94.3% and 88.6%, respectively) and the promoter methylation in PD-L2 decreased upon DAC treatment (90.7% and 83.6%, respectively). The relative mRNA expression levels of PD-L2 mRNA in Kasumi-1, SKNO-1-PGK, SKNO-1-siA/E and U937A/E cell lines treated with 2.5 μmol/L of DAC were significantly higher than those in control group (all P<0.05). Especially, PD-L2 expression increased more in cell lines silencing the AML1-ETO gene (SKNO-1-siA/E was 35.80% more compared to SKNO-1-PGK, P=0.031) and less in cell lines overexpressing the AML1-ETO gene (U937A/E + ZnSO₄ decreased 95.51% compared to U937A/E, P=0.036) after DAC treatment. PD-L2 mRNA expression increased in a concentration-dependent manner upon DAC treatment in Kasumi-1 and SKNO-1-PGK cell lines (Kasumi-1: r²=0.87; SKNO-1-PGK: r²=0.93, all P<0.05).
Conclusion The expression of PD-L2 in AML1-ETO-positive AML cells is regulated by DNA methylation and AML1-ETO might be involved in the inhibition of PD-L2 gene expression.

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