Asb2对急性髓系白血病干细胞和多能祖细胞增殖的影响及其作用机制

Effect of Asb2 on Proliferation of Stem Cells and Pluripotent Progenitor Cells in Acute Myeloid Leukemia and Its Potential Mechanism

目的:探讨锚蛋白重复序列和细胞信号抑制因子盒蛋白2(Asb2)对急性髓系白血病(AML)干细胞和多能祖细胞增殖的影响及其相关分子机制。方法:使用重组慢病毒构建上调或下调Asb2表达的人急性髓系白血病细胞株KG-1a,CCK-8方法检测细胞存活情况。Western blot检测AML干细胞和多能祖细胞及对照组细胞中Asb2、Jak2、Jak3、Foxo1、Mcl-1和β-catenin蛋白分子的表达。在NIH3T3细胞中改变Asb2、Skp2和β-Trcp的表达,Western blot检测细胞中Foxo1、Mcl-1和β-catenin蛋白表达。结果:CCK-8结果显示,上调KG-1a中Asb2表达能够明显抑制细胞增殖(P<0.01),而下调KG-1a中Asb2表达能够明显促进细胞增殖(P<0.01);相比较于对照组细胞,Asb2在AML干细胞和多能祖细胞中表达被抑制,而Jak2、Jak3、Foxo1、Mcl-1和β-catenin蛋白表达被加强;在NIH3T3细胞中,加强Asb2的表达会导致Foxo1、Mcl-1和β-catenin表达降低,Skp2或β-Trcp与Asb2的协同表达会进一步抑制Foxo1、Mcl-1和β-catenin的表达,而shRNA-Skp2或shRNA-β-Trcp的表达会增强Foxo1、Mcl-1和β-catenin的蛋白表达量。结论:Asb2可能通过下调Jak2、Jak3、Foxo1、Mcl-1和β-catenin蛋白分子的表达影响AML干细胞和多能祖细胞增殖,Asb2是急性早幼粒细胞白血病的潜在治疗靶点。

Objective:To investigate the effects of Asb2 expression on the proliferation of acute myeloid leukemia(AML)stem cells and progenitor cells as well as its related molecular mechanism.Method:Recombinant lentivirus was used to construct Asb2 down-regulation or up-regulation KG-1a cells.Proliferation of KG-1a cells was assayed by CCK-8 method.Western blot was employed to detect the expression of Asb2,Jak3,Jak2,Foxo1,Mcl-1 andβ-catenin in AML stem cells and progenitor cells,normal stem cells and progenitor cells as control.In NIH3T3 cells,western blot was employed to detect the expression of Foxo1,Mcl-1 andβ-catenin after changing the expression of Asb2,Skp2 orβ-Trcp.Results:The cell inhibition rate of Asb2 up-regulation group was significantly higher than that of empty vector group(P<0.01).The cell proliferation rate of the shRNA-Asb2 group was significantly higher than that of the shRNA-NC group(P<0.01).In contrast to normal cells,the expression of Asb2 was significantly inhibited in AML stem cells and progenitor cells whereas the expression of Jak3,Jak2,Foxo1,Mcl-1 andβ-catenin was significant elevated.In NIH3T3 cells,the over-expression of Asb2 could induce the degradation of Foxo1,Mcl-1andβ-catenin.The co-expression of Skp2orβ-Trcp with Asb2could furthur promote the degration of Foxo1,Mcl-1andβ-catenin,whereas the knockdown of Skp2orβ-Trcp could rescue Asb2-induced degradation of Foxo1,Mcl-1andβ-catenin.Conclusion:Asb2may affect the proliferation of AML stem cells and progenitor cells through inducing the degradation of Jak3,Jak2,Foxo1,Mcl-1andβ-catenin.Asb2is a potential therapeutic target for acute promyelocytic leukemia.