Xie W Q, Yang X, Gu R X, Tian Z, Xing H Y, Tang K J, Rao Q, Qiu S W, Wang M, Wang J X
State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China Tianjin Institutes of Health Science, Tianjin 301600, China.
Zhonghua Xue Ye Xue Za Zhi. 2023 May 14;44(5):366-372. doi: 10.3760/cma.j.issn.0253-2727.2023.05.003.
To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (<0.05) . This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.
通过建立诱导AML1-ETO（AE）融合基因表达的白血病细胞模型，研究AE融合基因对U937白血病细胞生物学功能的影响。利用慢病毒载体系统建立了U937细胞系（U937-AE）中AE融合基因的强力霉素（Dox）依赖性表达。分别采用细胞计数试剂盒8法（包括PI和西达苯胺诱导）检测细胞增殖、细胞周期诱导分化试验。利用转录组测序和代谢组测序初步探讨了AE融合基因对U937-AE细胞生物学功能的影响。①成功构建了Dox依赖性Tet-on调控系统，以调节U937-AE细胞中AE融合基因的稳定表达。②诱导AE融合基因表达24小时后，细胞增殖减慢，AE表达组的细胞增殖率（3.47±0.07）低于AE不表达组（3.86±0.05）（<0.05）。细胞周期中G(0)/G(1)期细胞比例增加，AE表达组[（63.45±3.10）%]高于AE不表达组[（41.36±9.56）%]（<0.05）。随着AE的表达，表达CD13和CD14的细胞比例下降。AE阴性组明显高于AE阳性组（<0.05）。③转录组测序基因集的富集分析显示，AE表达后，静止、活化B细胞核因子κ轻链增强子、干扰素-α/γ等炎症反应和免疫调节信号显著富集。④在AE的影响下，U937-AE细胞发生脂肪酸代谢紊乱。AE表达细胞中中短链脂肪酸酰基肉碱代谢产物浓度降低，如丙酰L-肉碱，其中AE表达组（0.46±0.13）低于AE不表达组（1.00±0.27）（<0.05）。AE表达细胞中一些长链脂肪酸酰基肉碱的代谢产物浓度升高，如十四烷酰肉碱，其中AE表达组（1.26±0.01）高于AE不表达组（1.00±0.05）（<0.05）。本研究成功建立了可诱导AE表达的白血病细胞模型。AE表达阻断细胞周期并抑制细胞分化。表达AE的U937-AE细胞中与炎症反应相关的基因集显著富集，且脂肪酸代谢紊乱。
