摘要
目的探讨缺失核定位信号的PML,即PML(NLS-)干扰对急性早幼粒细胞白血病细胞HL-60增殖与凋亡的影响。方法将靶向PML(NLS-)基因的3组干扰质粒pGpu6-PML(NLS-)shRNA和阴性对照质粒pGpu6-NCshRNA分别转染HL-60细胞,转染后48 h,G418筛选阳性克隆,分别命名为Si-1、Si-2、Si-3和NC组,并设空白对照组。采用RT-PCR法和Western blot法检测各组细胞中PML(NLS-)基因mRNA的转录水平和蛋白的表达水平;MTT法检测细胞的增殖活力;流式细胞术分析细胞的细胞周期及凋亡情况。结果 Si-1和Si-2组HL-60细胞PML(NLS-)基因mRNA的转录水平和蛋白的表达水平与空白对照组相比明显减低(P<0.05),有干扰效果;Si-1组HL-60细胞的增殖水平与空白对照组相比明显降低(P<0.05),以转染后48 h降低最为显著(P<0.01);抑制PML(NLS-)表达可引起HL-60细胞S期比例增高,G1和G2期比例下降(P<0.05);Si-1组细胞凋亡率明显高于NC组和空白对照组(P<0.05)。结论干扰PML(NLS-)的表达可促进HL-60细胞的凋亡,抑制其增殖。
Objective To investigate the effect of RNA interference targeting PML （NLS-） on proliferation and apoptosis of acute promyelocytic leukemia （APL） HL-60 cells. Methods HL-60 cells were transfected with three groups of interference plasmids pGpub-PML（NLS-） shRNA targeting PML（NLS-）, named as Si-1, Si-2 and Si-3, and negative control （NC） plasmid pGpub-NCshRNA respectively, using those untransfected as blank control. Positive clones were screened with G418 48 h after transfection, and determined for transcription level of PML （NLS-） mRNA by RT-PCR, for expression level of PML （NLS-） protein by Western blot, for proliferation activity by MTI＂ method, and for cell cycle and apoptosis by flow cytometry. Results Compared with those in blank control group, the transcription level of PML （NLS-） mRNA and expression level of PML（NLS-） protein in HL-60 cells in Si-1 and Si-2 groups decreased significantly （P 〈 0. 05）, indicating interfering effects. However, the proliferation level of HL-60 cells in Si-1 group, especially that 48 h after transfection （P 〈 0. 01）, was significantly lower than those in blank control group （P 〈 0. 05）. The inhibition of PML （NLS-） expression increased the percentage of HL-60 cells at S phase while decreased that at G1 and G2 phase （P 〈 0. 05）. The apoptosis rate of cells in Si-1 was significantly higher than those in NC and blank control groups （P 〈 0. 05）. Conclusion The interference of PML（NLS-） gene expression promoted the apoptosis and inhibited the proliferation of HL-60 cells.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第3期354-358,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金(81171658)
重庆市自然科学基金计划重点项目(2011BA5037)
