靶向沉默Eps8基因对人慢性髓系白血病K562细胞生物学活性的影响及其分子机制的探讨

Effect of Silencing Eps8 Gene Expression on the Biology Activity of Human Leukemia K562 Cells and Its Molecular Mechanism

目的:探索靶向沉默Eps8(表皮生长因子受体通路底物8)基因对人慢性髓系白血病K562细胞生物学活性的影响及其可能的分子机制。方法:采用慢病毒介导RNA干扰技术靶向沉默K562细胞中Eps8基因。实验分组为:空白对照(blank control)组、Eps8-shRNA靶向沉默(K562-shRNA)组和阴性对照(K562-NC)组。在荧光显微镜下观察转染效率;应用RT-PCR法和Western blot法分别从mRNA转录水平和蛋白水平验证Eps8基因沉默效率;台盼蓝拒染法和MTT法检测细胞体外增殖能力变化;流式细胞术检测细胞凋亡率变化;甲基纤维素克隆形成实验验证细胞集落形成能力;Western blot法检测沉默Eps8基因后AKT/mTOR及其下游信号通路磷酸化蛋白的表达情况。结果:成功建立稳定低表达Eps8的K562细胞株(K562-shRNA)和阴性对照细胞株(K562-NC)。与空白对照组和K562-NC组相比,沉默Eps8基因后K562-shRNA组细胞Eps8转录和蛋白表达水平均显著降低(P<0.01);体外培养48 h后,K562-shRNA组细胞增殖能力明显下降(P<0.05);K562-shRNA组细胞凋亡率为(5.81±0.39)%,空白对照组和K562-NC组细胞凋亡率分别为(1.02±0.70)%和(1.19±0.15)%,提示K562-shRNA组细胞凋亡率与空白对照组和K562-NC组之间有显著性差异(P<0.05),而空白对照组和K562-NC组之间没有显著性差异(P>0.05);体外甲基纤维素半固体培养10 d后,K562-shRNA组细胞集落形成能力与空白对照组和K562-NC组相比有显著性差异(P<0.05)。沉默Eps8基因可下调K562细胞p-AKT(308)和p-AKT(473)的表达(P<0.05),而t-AKT表达水平没有明显变化。另外,沉默Eps8基因可下调AKT下游的pmTOR和p-PRAS40蛋白的表达(P<0.05),而总mTOR和总PRAS40的表达水平没有明显变化。结论:利用慢病毒介导的RNA干扰技术靶向沉默Eps8基因能够抑制K562细胞增殖,促进其凋亡,其作用机制可能与AKT/mTOR及其下游信号通路下调有关。

Objective： The study was to investigate the effect of silencing Eps8 gene expression on proliferation and apoptosis of human leukemia K562 cells and its molecular mechanism. Methods： The expetriments were divided into 3 groups,including blank control group（ K562 cells without treatment）,K562-shRNA group（ K562 cells transfected by specific Eps8-shRNA lenticiral vector） and K562-NC group（ K562 cells transfected by negtive control lenticiral vector）. K562 cells with stably-silenced Eps8 gene were constucted by lentibirus-mediated RNA technology. The efficacy of transfection was observed by fluorescence microscopy and the changes of Eps8 mRNA and protein level were detected by RT-PCR and Western blot respectively. Cell proliferation was confirmed by typan blue exclusion and MTT method. The apoptosis rate of cells was analysed by the flowcytometry, and colony forming was detected by methylcellulose colony forming assay. The protein level change of phosphrylated-AKT were detected by Western blot.Results： Stably-silenced Eps8 gene K562 cells and the negative control cells were successfully constructed. Compared with the blank control group and the K562-NC group,the proliferation of K562-shRNA cells were siginificantly inhibited（ P〈0. 05）; the apoptosis of K562-shRNA cells increased（ P〈0. 05）. In addition,the methylcellulose colony forming assay showed that the colony forming was dramatically suppressed in K562-shRNA cells（ P〈0. 05）.Furthermore,knocking down Eps8 gene reduced the protein level of AKT phosphrylation at both residue Ser437 and Thr308（ P〈0. 05）,while there was no obvious change in the level of total-AKT（ P〉0. 05）. Knocking downEps8 gene reduced the protein level of m-TOR phosphrylation and PRAS40 phosphrylation（ P〈0. 05）,while there was no obvious change in the level of total-m TOR and PRAS40（ P〉0. 05）. Conclusion： Silencing Eps8 gene through lentvirus can inhibit the cell proliferation and promote the apoptosis of human leukemia K562 cells,which possibly relates with the inhibition of AKT/mTOR activation.