Sp1基因通过CD59调控T细胞急性淋巴细胞白血病细胞增殖和凋亡

Sp1 gene regulates proliferation and apoptosis of T cell acute lymphoblastic leukemia cells through CD59

目的探究Sp1和CD59在T细胞急性淋巴细胞白血病(T-ALL)中的表达模式,以及Sp1和CD59对T-ALL细胞功能的影响。方法通过实时荧光定量聚合酶链反应(qRT-PCR)检测了20例T-ALL患者和20名健康受试者的骨髓样本,以及人正常外周血单个核细胞(PBMC)和T-ALL细胞系CCRF-CEM中Sp1和CD59 mRNA表达水平。两种细胞均购自美国典型培养物保藏中心(ATCC)。CCRF-CEM细胞分为6组:对照组(未处理的细胞)、si-NC组(转染阴性对照siRNA的细胞)、si-Sp1组(转染靶向Sp1的siRNA的细胞)、pcDNA3.1-NC组(转染阴性pcDNA3.1质粒的细胞)、pcDNA3.1-CD59组(转染过表达CD59的pcDNA3.1质粒的细胞)和si-Sp1+pcDNA3.1-CD59组(同时转染靶向Sp1的siRNA和过表达CD59的pcDNA3.1质粒的细胞)。通过细胞计数试剂盒8(CCK-8)测定细胞增殖,采用流式细胞仪检测细胞凋亡,采用伤口愈合实验检测细胞迁移,通过Transwell实验检测细胞侵袭,采用蛋白质印迹分析(Western blot)检测PI3K/AKT/GSK3β信号通路相关分子的蛋白表达。结果T-ALL患者骨髓中Sp1和CD59的mRNA表达水平均显著高于健康人(P<0.05)。CCRF-CEM细胞中的Sp1和CD59的mRNA表达水平显著高于PBMC(P<0.05)。与对照组相比,si-Sp1组CD59 mRNA和蛋白表达水平显著降低(P<0.05)。对照组和pcDNA3.1-CD59组中Sp1的mRNA和蛋白表达水平无显著差异(P>0.05)。与对照组相比,si-Sp1组细胞活力、迁移和侵袭能力显著降低,而pcDNA3.1-CD59组显著升高(P<0.05);si-Sp1组细胞凋亡率显著升高,而pcDNA3.1-CD59组显著降低(P<0.05);si-Sp1组的p-PI3K、p-AKT和p-GSK3β蛋白表达水平显著降低,而pcDNA3.1-CD59组显著升高(均P<0.05)。与pcDNA3.1-CD59组相比,si-Sp1+pcDNA3.1-CD59组的细胞活力、迁移和侵袭能力显著降低,细胞凋亡率显著升高,p-PI3K、p-AKT和p-GSK3β蛋白表达水平显著降低(均P<0.05)。结论Sp1和CD59在T-ALL中异常高表达。沉默Sp1通过靶向下调CD59的表达来抑制T-ALL细胞增殖、迁移和侵袭及PI3K/AKT/GSK3β信号通路活化,并促进细胞凋亡。

Objective To investigate the expression patterns of Sp1 and CD59 in T-cell acute lymphoblastic leukemia(T-ALL)and the effects of Sp1 and CD59 on the function of T-ALL cells.Methods The mRNA expression levels of Sp1 and CD59 were detected in bone marrow samples from 20 T-ALL patients and 20 healthy subjects and in peripheral blood mononuclear cells(PBMC)and T-ALL cell lines(CCRF-CEM)by quantitative real-time polymerase chain reaction(qRT-PCR).Both PBMC and CCRF-CEM cells were purchased from the American Type Culture Collection(ATCC).CCRF-CEM cells were divided into 6 groups:control group(no treatment),si-NC group(transfected with negative control siRNA),si-Sp1 group(transfected with siRNA targeting Sp1),pcDNA3.1-NC group(transfected with negative pcDNA3.1 plasmid),pcDNA3.1-CD59 group(transfected with pcDNA3.1 plasmid overexpressing CD59)and si-Sp1+pcDNA3.1-CD59 group(transfected with siRNA targeting Sp1 and pcDNA3.1 plasmid overexpressing CD59).CCK-8 was used to detect the cell proliferation,flow cytometry was used to detect the cell apoptosis,wound healing experiment was used to detect the cell migration,Transwell experiment was used to detect the cell invasion,and Western blot was used to detect the protein expression of PI3K,AKT,related molecules in GSK3βsignaling pathway.Results The mRNA expression levels of Sp1 and CD59 in bone marrow of T-ALL patients were significantly higher than those of healthy controls(P<0.05).In addition,the mRNA expression levels of Sp1 and CD59 in CCRF-CEM cells were significantly higher than that of PBMC(P<0.05).Compared with control group,the mRNA and protein expression levels of CD59 in si-Sp1 group were significantly reduced(P<0.05).There was no significant difference in Sp1 mRNA and protein expression levels between control group and pcDNA3.1-CD59 group(P>0.05).Compared with control group,the cell viability and the migration and invasion abilities were significantly reduced in si-Sp1 group,while they were significantly increased in pcDNA3.1-CD59 group(all P<0.05).Compared with control group,the apoptosis rate was significantly increased in si-Sp1 group,but significantly reduced in pcDNA3.1-CD59 group(all P<0.05).Compared with control group,the p-PI3K,p-AKT,and p-GSK3βprotein expression levels were significantly reduced in si-Sp1 group,but significantly increased in pcDNA3.1-CD59 group(all P<0.05).Compared with pcDNA3.1-CD59 group,the cell viability and the migration and invasion abilities were significantly reduced in si-Sp1+pcDNA3.1-CD59 group,the apoptosis rate was significantly increased,while the protein expression levels of p-PI3K,p-AKT and p-GSK3βwere significantly reduced(all P<0.05).Conclusion Sp1 and CD59 are abnormally highly expressed in T-ALL.Silencing Sp1 may inhibit the cell proliferation,migration and invasion,and the activation of PI3K/AKT/GSK3βsignaling pathway,and promote the apoptosis by targeting down-regulation of CD59 expression in T-ALL cells.