YB-1蛋白对白血病细胞K562/A02生物学行为的影响

Influence of YB-1 Protein on the Biological Behaviour in K562/A02 Cells

本研究探讨YB-1基因表达下调后对白血病细胞K562/A02生物学行为的影响及其机制。采用脂质体介导的方法将已构建的人YB-1基因特异性shRNA及随机片段HK的真核表达载体转染进白血病细胞K562/A02中,RT-PCR和免疫印迹反应检测YB-1 mRNA和蛋白表达量的改变;采用MTT法和细胞周期测定分析对白血病细胞增殖的影响;用AnnexinⅤ检测白血病细胞的凋亡程度;采用MTT法检测细胞IC50值变化;RT-PCR和流式细胞术检测基因转染前后细胞中的MDR1 mRNA和P-gp表达的变化。结果表明,阳性转染的3组细胞YB-1基因和蛋白的表达量明显低于随机片段组和未转染细胞;生长曲线提示阳性转染组细胞生长缓慢,细胞周期动力学分析显示阳性转染组G1期细胞增多,G2期与S期细胞显著减少;在小剂量As2O3作用下,阳性转染组细胞的凋亡数量明显高于对照组细胞;阳性转染细胞的阿霉素IC50明显低于随机片段组和阴性对照组;阳性转染细胞MDR1 mRNA水平明显降低,P-gp的峰值较对照组明显左移。结论:YB-1特异性shRNA能有效降低白血病细胞中YB-1的表达,减慢细胞生长,增加白血病细胞对化疗药物的敏感性,加速细胞凋亡。

The aim of this study was to investigate whether the growth,apoptosis and sensitivity to anticancer agent could be altered after introduction of YB-1 shRNA eukaryotic expression vector into the K562/A02 cells,and its possible molecular mechanisms.The recombinant eukaryotic expression plasmids including YB-1 shRNA and the vector-random-sequence were introduced into K562/A02 cells by lipofectamine mediation,and the positive clones were screened by G418.RT-PCR and Western blot were employed to detect the expression of mRNA and protein of YB-1 in leukemia cells,respectively.The proliferative ability of the cells was determined by MTT assay and cell cycle analysis.Apoptosis of K562/A02 cells was assayed by AnnexinⅤ-FITC/PI double labeled flow cytometry.The drug sensitivity to anticancer agent was determined by MTT assay.The expressions of MDR1 gene and P-gp were detected by RT-PCR and flow cytometry respectively.The results indicated that the levels of mRNA and protein of YB-1 decreased dramatically in three groups of positively transfected cells when compared with control cells.The inhibitory rates of 3 different shRNA sequences targeting YB-1 gene were（65.1±2.1）%,（27.4±1.3）% and（67.4±1.6）% respectively.The introduction of exogenous YB-1 shRNA gene into K562/A02 cells resulted in decreased levels of the proliferative ability in K562/A02 cells,and displayed higher at G1,lower at G2 and S phase in cell cycle distribution in comparison with the control groups.AnnexinⅤ/PI detection indicated higher AnnexinⅤ＋ ratio in 3 groups of positively transfected cells after being treated with As2O3 of 0.5 μmol/L for 24 hours.The IC50 values of doxorubicin in 3 groups of positively transfected cells were significantly lower than that in control group.The level of MDR1 gene and P-gp decreased significantly in 3 groups of positively transfected cells.It is concluded that the transfection with YB-1 shRNA gene can inhibit the proliferation of leukemia cells and induce cell apoptosis.The expression of MDR1 mRNA and P-gp decrease after transfection of YB-1 shRNA into K562/A02 cells.