沉默SET基因对急性早幼粒白血病NB4-R1细胞的作用

Effect of Silencing SET Gene on Acute Promyelocytic Leukemia NB4-R1 Cells

目的:探讨沉默SET基因对耐维甲酸急性早幼粒白血病NB4-R1细胞生物学特性的影响。方法:将含SET基因特异shRNA干扰序列的慢病毒载体pGCSIL,与其他包装质粒在293T细胞中包装成具有感染性的慢病毒质颗粒。收集并浓缩慢病毒,转染体外培养的NB4-R1细胞并建立稳定转染的细胞株。用荧光实时定量PCR和蛋白凝胶电泳Western blot验证转染后SET基因在NB4-R1细胞中的干扰效率,并检测沉默SET基因对蛋白磷酸酶PP2A表达的影响。用流式细胞术分析沉默SET基因前后NB4-R1细胞周期的变化。结果:与对照组相比,实验组SET基因的表达被有效抑制,PP2A表达增高。沉默SET基因导致NB4-R1细胞G_0/G_1期明显增加,S期细胞比例明显减少,差异具有统计学意义(P<0.05)。结论:利用慢病毒靶向干扰SET基因的表达可以使PP2A表达增高,并扰乱NB4-R1细胞的增殖周期。本研究为进一步研究SET相关的急性早幼粒细胞白血病临床靶向治疗奠定实验基础。

Objective:To investigate the effect of silencing SET gene on the biological characteristics of acute promyelocytic leukemia NB4-R1 cells.Methods:The expression vector of pGCSIL containing SET- shRNA were transfected into 293 T cells by using other packaging plasmids.The supernatant of the 293 T cells was harvested for lentivirus.The SET- shRNA lentiviral vector was transfected into acute promyelocytic leukemia NB4-R1 cells and a stably transfected cell line was established.Real-time quantitative PCR and Western blot were used to assay the silencing efficiency on SET gene and the expression of PP2 A.The cell cycle distribution was tested by flow cytometry.Results:The expression of SET in experimental group statistically decreased as compared with that of the control group.The expression of PP2 A was obviously raised at the level of mRNA and protein.The percentage of NB4-R1 cells in G0/G1phase significantly increased,while the percentage of cells in S phase significantly decreased.Conclusion:The silencing gene in acute promyelocytic leukemia NB4-R1 cells using SET- shRNA lentiviral vector can increase the expression of PP2 A and interfere of the cell cycle in NB4-R1 cells.This study has laid a experimental base for targed therapy of patients with acute promyelocytic leukemia.