过表达TRAF6对人急性髓系白血病细胞自噬活性的影响

该文旨在探讨过表达肿瘤坏死因子受体相关因子6(tumor necrosis factor receptorassociated factor 6,TRAF6)对人急性髓系白血病(acute myeloid leukemia,AML)细胞自噬活性的影响。利用基因表达数据库GEO分析TRAF6在AML患者白血病细胞中的mRNA表达水平。通过癌症基因组图谱TCGA分析TRAF6表达与AML患者临床预后的关系。将TRAF6重组质粒载体转染人AML细胞系(KG-1a和THP-1),采用自噬激活剂雷帕霉素(Rapamycin)和自噬相关抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)、巴弗洛霉素A1(bafilomycin A1,Baf-A1)分别处理AML细胞。荧光定量PCR、蛋白免疫印迹技术检测过表达TRAF6后白血病细胞自噬标志物(LC3和p62)mRNA和蛋白水平;免疫荧光方法检测LC3绿色荧光斑点结构(puncta);流式细胞术检测细胞凋亡率;CCK-8实验检测AML细胞的体外增殖能力。结果显示,AML患者白血病细胞高表达TRAF6(P<0.01);TRAF6高表达的白血病患者总体生存率和无事件生存率均较TRAF6低表达组显著降低(P=0.01)。TRAF6重组质粒转染能够显著增加两株AML细胞系中TRAF6的mRNA和蛋白水平(P<0.05)。Rapamycin处理能够激活AML细胞系自噬水平,过表达TRAF6后AML细胞LC3 mRNA和LC3II蛋白水平表达上调(P<0.05)、p62 mRNA和蛋白水平下调(P<0.05)以及LC3 puncta聚集增多。用Baf-A1处理以阻断过表达TRAF6的白血病细胞系中的自噬流后,LC3II蛋白表达水平显著提高(P<0.05)。3-MA处理过表达TRAF6的白血病细胞后,LC3II蛋白表达减少、p62蛋白表达增加(P<0.05)。此外,过表达TRAF6降低白血病细胞凋亡率和促进细胞的体外增殖(P<0.001),而过表达TRAF6后联合3-MA处理则可逆转TRAF6对白血病细胞的抗凋亡和促增殖作用(P<0.001)。以上研究结果提示,过表达TRAF6能够增强AML细胞的自噬活性,促进AML细胞的生长。

Effects of TRAF6 Over-Expression on Autophagic Activity of Human Acute Myeloid Leukemia Cells

This work was to investigate the effects of TRAF6(tumor necrosis factor receptor-associated factor 6)overexpression on autophagic activity of human AML(acute myeloid leukemia)cells.The expression levels of TRAF6 mRNA in AML patients derived from GEO(gene expression omnibus)database were analyzed.The relationship between the expression of TRAF6 and the clinical prognosis of AML patients were explored based on TCGA(The Cancer Genome Atlas)database.The TRAF6 recombination plasmids were transfected into two human acute myeloid leukemia cell lines(KG-1a and THP-1).Autophagy activator(Rapamycin)and two different autophagy inhibitors,3-MA(3-methyladenine)and Baf-A1(bafilomycin A1)were used to treat the leukemic cells.The effects of TRAF6 overexpression on the mRNA and protein levels of autophagic markers LC3 and p62 in leukemic cells were determined by qRT-PCR and Western blot techniques,respectively.The LC3 puncta was determined by immunofluorescence.The apoptosis rate was detected by flow cytometry.The cell proliferation activity in vitro was evaluated by CCK-8 assay.The results showed that the expression levels of TRAF6 mRNA in AML patients were higher than those observed in normal controls(P<0.01).Additionally,high expression of TRAF6 showed a trend towards lower OS(overall survival)and EFS(event-free survival)(P=0.01)in AML patients,than that in low expression of TRAF6 group.The expression levels of TRAF6 mRNA and protein were significantly increased in TRAF6 overexpression group compared with those in the control group(P<0.05).Rapamycin treatment enhanced the autophagic activity of leukemic cells.Importantly,TRAF6 overexpression significantly upregulated LC3 mRNA and LC3II protein levels(P<0.05),downregulated p62 mRNA and protein levels and increased the accumulation of LC3 puncta in leukemic cells(P<0.05).Notably,Baf-A1 treatment significantly increased LC3II protein levels in TRAF6-enforced cells(P<0.05).Exposure to 3-MA significantly downregulated LC3II protein levels and upregulated p62 protein levels in TRAF6-enforced expression cells(P<0.05).Finally,the apoptosis rate was significantly decreased and the cell proliferation was significantly enhanced in TRAF6-enforced group(P<0.001),while the treatment of 3-MA attenuated the ability of TRAF6 overexpression mediated growth advantage(P<0.001).Abovementioned results suggest that TRAF6 high expression enhances the autophagic activity and promotes leukemic cell growth.