miR-34b调控去泛素化酶USP22在急性髓细胞白血病细胞HL60中的生物学作用

Suppressive Function of miR-34b on Deubiquitinase USP22 in Acute Myeloid Leukemia HL60 Cells

目的研究miR-34b调控其靶基因USP22分子机制及其在急性髓细胞白血病细胞HL60中的生物学效应。方法荧光定量PCR (qPCR)检测miR-34b、USP22转录水平;蛋白免疫印迹(Western blotting)检测USP22、Bax、Bcl-2蛋白表达水平;急性髓细胞白血病HL60细胞中过表达、抑制miR-34b或干扰USP22后,CCK-8及AnneximV分别检测细胞增殖活性及凋亡变化。双荧光素酶报告基因系统证实miR-34b与USP22的3,非编码区(3-UTR)相互作用。结果 miR-34b在白血病细胞HL60中表达下调,为外周血健康对照单个核细胞的0. 46 ±0. 07倍(P<0.01)。与对照细胞相比,在HL60细胞中过表达miR-34b或抑制miR-34b后,细胞相对活性分别出现减低或增强现象(P<0.01)。miR-34b在HL60中过表达后,凋亡细胞(29.91 ±1. 14)%较对照细胞(8.77 ±0. 23)%增加了 2.41倍(P<0.01), Western印迹检测证实了促凋亡分子Bax及抑制凋亡分子Bcl-2在miR-34b过表达后分别出现了上调及下调现象。利用在线预测软件预测USP22可能是miR-34b直接调控基因。在HL60细胞中过表达或抑制miR-34b后,USP22转录水平及蛋白表达水平均不同程度上调及下调(P<0.01)。双荧光素酶报告基因系统证实miR-34b过表达后,USP22野生型的3-UTR较对照组显著下调(P<0.01),而突变型无显著改变。进一步敲减USP22后HL60细胞增殖活性减低,并诱导细胞凋亡。结论 miR-34b对白血病细胞HL60的抑癌活性可部分通过抑制USP22得以实现;同时过表达miR-34b或抑制USP22表达有望成为急性髓细胞白血病的诊治靶点。

Objective To investigate the role of miR-34b and its target USP22 in acute myeloid leukemia ( AML) cells HL60. Methods qPCR was used to measure transcriptional level of miR-34b and USP22. The protein level of USP22 , Bax and Bcl-2 was evaluated by Western blotting assay. The AML cells HL60 was treated with miR-34b mimics, inhibitors or USP22 siRNA and subsequently subjected to proliferation and apoptosis assessment by CCK-8 and Annexin.V methods. Dual luciferase report assay was performed to confirm the direct interaction between miR-34b and USP22 3'UTR. Results The expression of miR-34b was down-regulated in HL60 cells, compared to control cells N0M01 ( with 0. 46 ± 0. 07 fold change, P < 0. 01 ). Overexpression or inhibition of miR-34b in HL60 led to increased or decreased proliferation rate, respectively (P <0. 01). In addition ,overexpression of miR-34b in HL60 induced apoptosis when compared to control cells [(29. 91 ±1. 14)% vs,(8. 77 ±0. 23)%, with 2. 41 fold increase, P <0. 01)]. Bax was upregulated while Bcl-2 wareduced when HL60 cells were treated with miR-34 mimics. Through online prediction tool, we identified USP22 as potential target of miR-34b. Overexpression of miR-34b upregulated USP22 , while inhibition of miR-34b down-regulated USP22 ( P < 0. 01). Dual luciferase report system confirmed that the luciferase activity of plasmid containing wild type of USP22 3'UTR was significantly inhibited when HL60 cells were treated with miR-34b mimics ( P = 0. 0007 ), while no change was observed in the report system with mutant type of USP22 3'UTR. Furthermore, knockdown of USP22 in HL60 cells resulted in reduced proliferate rate and induced apoptosis. Conclusion The miR-34b exerts its tumor suppressive effect partly through USP22. Overexpression of miR-34b or inhibition of USP22 may be promising targets for the treatment of AML.