硼替佐米对急性髓性白血病细胞凋亡及SALL4基因和Wnt/β-catenin信号通路的影响

Influence of Bortezomib on apoptosis of acute myelogenous leukemia cells as well as on SALL4 gene and Wnt/β-catenin signaling pathway

目的探讨硼替佐米(Bortezomi,Bor)对急性早幼粒细胞白血病NB4细胞株和急性红白血病TF1细胞株增殖、凋亡及SALL4基因和Wnt/β-catenin信号通路下游靶基因C-myc、CCDN1表达的影响。方法分别用10、30、50 nmol/L的Bor处理NB4和TF1细胞12、24、48 h,并设对照组(不加Bor)。采用MTT法检测细胞的增殖活力;流式细胞术检测细胞凋亡率;RT-PCR和荧光定量PCR法检测细胞中SALL4、C-myc和CCDN1基因的表达。结果 Bor可抑制两种细胞的增殖,且呈时间和剂量依赖性,NB4和TF1细胞经Bor处理48 h的IC50值分别为23.97和25.36 nmol/L。Bor可诱导两种细胞凋亡,且呈剂量依赖性,经30和50 nmol/L Bor处理24 h的NB4细胞的凋亡率与对照组相比,差异有统计学意义(P<0.05);经50 nmol/L Bor处理24 h的TF1细胞的凋亡率与对照组相比,差异有统计学意义(P<0.05)。Bor可显著抑制两种细胞中SALL4基因及C-myc、CCDN1基因的表达。经50 nmol/L Bor作用24 h后,两种细胞中3种基因的表达与对照组相比,差异均有统计学意义(P<0.05)。SALL4基因与C-myc和CCDN1基因的表达明显相关(rs值分别为0.857和0.929,P均<0.01)。结论 SALL4基因及Wnt/β-catenin信号通路下游靶基因C-myc和CCDN1基因表达的抑制在Bor诱导NB4和TF1细胞凋亡的过程可能起重要作用。

Objective To investigate the influence of Bortezomib （Bor） on proliferation and apoptosis of acute promyelocyte leukemia NB4 cell strain and acute erythroleukemia TF1 cell strain as well as on expressions of SALL4 gene and C-mye and CCND1 genes downstream in Wnt/β-catenin signaling pathway. Methods NB4 and TF1 cells were treated with 10, 30 and 50 nmol/L Bor for 12, 24 and 48 h separately, using those untreated as control, then determined for proliferation activity by MT]＂ method, for apoptosis rate by flow cytometry, and for expressions of SALL4, C-mye and CCND1 genes by RT-PCR and fluorescent quantitative PCR. Results Bor showed time- and dose-dependent inhibitory effect on proliferation of NB4 and TF1 cells, with ICso values of 23.97 and 25.36 nmol / L respectively 48 h after treatment. Bor induced the apoptosis of two kinds of cells in dose-dependent mode. The apoptosis rates of NB4 cells 24 after treatment with 30 and 50 nmol/L Bor and TF1 cells 24 h after treatment with 50 nmol/L Bor showed significant difference with those in control group （both P 〈 0. 05）. Bor inhibited the expressions of SALL4, C-mye and CCND1 genes in the two kinds of cells significantly. Twenty-four hours after treatment with 50 nmol/L Bor, the expression levels of the three genes in both NB4 and TF1 cells were significantly higher than those in control group （each P 〈 0. 05）. The expression of SALL4 gene was closed related to those of C-mye and CCND1 genes （rn = 0. 857, 0. 929, both P 〈 0. 01 ）. Conclusion The inhibition of SALL4 gene as well as target genes C-mye and CCND1 downstream in Wnt / β-catenin signaling pathway may play an important role in induction of apoptosis of NB4 and TF1 cells.