干扰GINS2表达对HL60细胞增殖和凋亡的影响

The Effect of Down-regulation of GINS2 on Proliferation and Apoptosis of HL60 Cells

目的:探讨靶向抑制GINS2基因表达后对人早幼粒细胞白血病细胞株HL60增殖和凋亡的影响及其机制。方法:RT-PCR分别检测GINS2基因在三株白血病细胞HL60、U937、THP-1的表达。设计与合成针对GINS2基因的干扰序列,稳定转染高表达该基因的HL60细胞,荧光显微镜观察绿色荧光蛋白的表达情况;RT-PCR和Western blot分别检测各组细胞转染后mRNA和蛋白质水平;MTT法检细胞的增殖和凋亡情况;流式细胞术分析细胞的凋亡率;Western blot检测凋亡相关蛋白的表达。结果:在三株白血病细胞株中,GINS2的表达量由低到高依次是:THP-1,U937,HL60。干扰质粒转染HL60细胞后,与阴性对照组及未处理组相比,干扰组GINS2的mRNA和蛋白质水平均显著降低,且细胞的增殖能力明显受抑。同时,凋亡相关蛋白Bax表达水平显著增高而Bcl2显著降低。结论:干扰GINS2基因表达后,其mRNA和蛋白质水平均显著降低,可通过上调Bax表达,下调Bcl2表达来抑制HL60细胞增殖并促进其凋亡。

Objective： To investigate the effect and mechanism of small interfering RNA targeted GINS2 expression on proliferation and apoptosis of human acute promyelocytic leukemia HL60 cells. Method： The expression of GINS2 complexes comprised in three leukemia cell lines HL60 ,U937,THP-1 were detected by Reversed-Transcript PCR（RT-PCR）.The plasmid specific targeted GINS2 expression were stably transfected into HL60 cells with high expression of this gene by means of lipidosome-mediated method. Then,the transcription and translation level were detected by RT-PCR and Western blot respectively. Meanwhile cell proliferation and apoptosis after transfection were measured by MTT method and flow cytometry. In addition,the change of apoptin Bax and Bcl2 were disclosed by western blot. Results Among three leukemia cell lines ,the expression of GINS2 from low to high was following as：THP-1,U937,HL60. After siRNA plasmid were transfected into HL60 cells,both GINS2 expression level of mRNA and protein in interfering group ,measuring by RT-PCR and Western blot method ,were down-regulated after transfection when compared with untreated control group and negative control group. Together, MTT and flow cytometry showed that cell growth was significantly inhibited.（P＜0.05）. Moreover,the expression lever of Bax was significantly increased whereas Bcl2 was dramatically decreased in positive transfected group.Conclusion： The down-regulation of mRNA and protein level of GINS2 complexes might induce apoptosis and inhibit cell growth by ways of up-regulation of Bax and down-regulation of Bcl2 in HL60 cell lines.