Bmi-1-siRNA通过PTEN/pAKT通路调控K562细胞体内外的增殖能力

Bmi-1-siRNA Regulates the Proliferation of K562 Leukemia Cells in vitro and in vivo by PTEN/pAKT Pathway

目的:观察Bmi-1基因沉默对白血病K562细胞体内外增殖能力的影响并初步探究二者之间的分子机制是否与PTEN/pAKT通路有关。方法:将Bmi-1小干扰RNA(siRNA)序列转染到K562细胞中降低其Bmi-1的表达;应用MTT比色法和软琼脂集落形成实验检测Bmi-1-siRNA对K562细胞体外增殖的影响;裸鼠皮下接种各组细胞,观察Bmi-1-siRNA对K562细胞在裸鼠体内的致瘤能力的影响;应用Western blot检测Bmi-1、PTEN、p-AKT等蛋白的表达。结果:Bmi-1-siRNA有效沉默了Bmi-1基因mRNA和蛋白的表达;沉默Bmi-1基因的表达能够抑制K562细胞的增殖活性、集落形成及裸鼠成瘤能力;Bmi-1基因沉默后,干扰组PTEN表达明显升高,而p-AKT活性明显下降;p-AKT抑制剂LY294002处理K562细胞后,p-AKT表达降低,集落形成及裸鼠成瘤能力相比未处理K562细胞降低;PTEN抑制剂Bpv处理K562-S1细胞后,PTEN的表达降低,而p-AKT、集落形成及裸鼠成瘤能力得以重塑。结论:Bmi-1基因有可能参与了对白血病细胞K562的体内外增殖能力的调控,PTEN/pAKT信号通路可能是介导这一调控过程的分子机制之一。

Objective:To investigate the effect of Bmi-1 gene silence on the proliferation ability of K562 cells in vitro and in vivo,and to explore the relation of molecular mechanism between proliferation ability of K562 cells in vitro and in vivo with PTEN/pAKT signaling pathway.Methods:The Bmi-1 small interference RNA(siRNA)sequences were transfected into K562 cells for decreasing Bmi-1 expression.The effect of Bmi-1 siRNA on the proliferation of K562 cells in vitro and in vivo was detected by MTT method and colony-forming test,the effect of Bmi-1 siRNA on the tumorogenicity of K562 cells was observed by subcutaneous inoculation of K562 cells,LY294002 and Bpv treated K562 cells in nude mice,the expression of Bmi-1,PTEN and pAKT proteins were detected by Western blot.Results:The Bmi-1 siRNA could inhibit the proliferation activity,colony-forming and tumor-forming abilities of K562 cells.After the silence of Bmi-1 gene,the PTEN expression in Bmi-1 gene-silenced group was significantly enhanced.While the pAKT expression in Bmi-1 gene-silenced group was significantly reduced;after the K562 cells were treated with LY294002(an inhibitor of pAKT),the pAKT expression colony-forming and tumor forming abilities were reduced in comparison with untreated K562 cells;after the K562-S1 cells were treated with Bpv(an inhibitor of PTEN),the PTEN expression decreased,while the pAKT expression,colony forming and tumor-forming abilities were restored.Conclusion:The Bmi-1 gene possibly involves in regulation of K562 proliferation in vivo and in vitro,the effect of PTEN/pAKT signaling pathway maybe one of molecular mechanisms mediating this regulation.