IRAK1/TRAF6通路在急性髓系白血病发生发展中的作用机制研究

Role of IRAK1/TRAF6 pathway in the occurrence and development of acute myeloid leukemia

目的探讨白细胞介素-1受体相关激酶(IRAK1)/肿瘤坏死因子受体相关因子6(TRAF6)通路影响急性髓系白血病(AML)发生发展的作用机制。方法选取54例AML患者为研究对象(AML组),30例于本院治疗的缺铁性贫血患者作为对照组,分离所有受试者骨髓单核细胞;体外常规培养人AML细胞系HL-60、KG-1、U937和人正常单核细胞系THP-1;将HL-60细胞分为空白组(转染Lipofectamine 3000试剂)、阴性对照(NC)组(转染空质粒)、IRAK1短发夹RNA(IRAK1 shRNA)组(转染IRAK1 shRNA);实时荧光定量PCR(qPCR)法检测单核细胞及不同细胞系各组中IRAK1、TRAF6 mRNA表达水平;CCK-8法检测各组细胞增殖抑制率;流式细胞术检测各组细胞凋亡率;蛋白免疫印迹法检测各组细胞β-连环蛋白(β-catenin)、细胞周期蛋白D1(CyclinD1)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)及IRAK1/TRAF6通路蛋白表达水平。结果与对照组比较,AML组单核细胞中IRAK1、TRAF6 mRNA表达水平显著升高(P<0.05)。与THP-1细胞比较,KG-1、U937、HL-60细胞中IRAK1、TRAF6 mRNA表达水平显著升高,且HL-60细胞最高(P<0.05)。与空白组和NC组比较,IRAK1 shRNA组细胞增殖抑制率、凋亡率及Bax蛋白表达显著升高,IRAK1 mRNA、TRAF6 mRNA、β-catenin、CyclinD1、Bcl-2、磷酸化-IRAK1(p-IRAK1)/IRAK1、TRAF6、核因子-кB(NF-кB)蛋白表达显著降低(P<0.05)。结论IRAK1/TRAF6通路与AML的发生发展有关,抑制IRAK1/TRAF6通路可抑制人AML细胞系HL-60增殖,促进其凋亡,IRAK1/TRAF6可能是AML治疗的潜在靶点。

Objective To investigate the mechanism of interleukin-1 receptor-associated kinase 1(IRAK1)/tumor necrosis factor receptor associated factor 6(TRAF6)pathway on the occurrence and development of acute myeloid leukemia(AML).Methods Fifty-four patients with AML were selected as the research objects(the AML group),another 30 patients with iron deficiency anemia treated in our hospital were used as the control group.Bone marrow monocytes were isolated from all subjects.Human AML cell lines HL-60,KG-1,U937 and human normal monocyte line THP-1 were cultured in vitro.HL-60 cells were divided into the blank group(transfection Lipofectamine 3000 reagent),the negative control(NC)group(transfection empty plasmid)and the IRAK1 short hairpin RNA(IRAK1 shRNA)group(transfection IRAK1 shRNA).The expression levels of IRAK1 mRNA and TRAF6 mRNA in monocytes were detected by real-time fluorescence quantitative PCR.CCK-8 assay was used to detect the proliferation inhibition rate of HL-60 cells.Flow cytometry was used to detect the apoptosis rate.Western blot assay was used to detect the expression levels ofβ-catenin,CyclinD1,antiapoptotic protein B-cell lymphoma-2(Bcl-2),apoptosis protein Bcl-2 related X protein(Bax)and IRAK1/TRAF6 pathway protein.Results Compared with the control group,the expression levels of IRAK1 mRNA and TRAF6 mRNA were significantly higher in the AML group(P<0.05).Compared with those in THP-1 cells,the expression levels of IRAK1 mRNA and TRAF6 mRNA were significantly higher in KG-1,U937 and HL-60 cells,and the HL-60 cells were the highest(P<0.05).Compared with those in the blank group and the NC group,the inhibition rate of cell proliferation,apoptosis rate and Bax protein expression were significantly higher in the IRAK1 shRNA group,and the expression levels of IRAK1 mRNA,TRAF6 mRNA,β-catenin,CyclinD1,Bcl-2,phosphorylation-IRAK1(p-IRAK1)/IRAK1,TRAF6 and nuclear factor-кB(NF-кB)protein expression were significantly lower(P<0.05).Conclusion IRAK1/TRAF6 pathway is related to the occurrence and development of AML.The inhibition of IRAK1/TRAF6 pathway can inhibit the proliferation and promote apoptosis of human AML cell line HL-60.IRAK1/TRAF6 pathway may be a potential target for AML treatment.