shRNA干扰AKT基因对Jeko-1细胞增殖、凋亡及Notch1信号通路的影响

Effects of Silencing AKT Gene by shRNA on Proliferation,Apoptosis and Notchl Signal Pathway in Jeko-1 Cells

目的:探讨RNA干扰沉默AKT基因对套细胞淋巴瘤Jeko-1细胞株的增殖、凋亡及Notch1信号通路的影响。方法:设计针对AKT基因短发夹RNA,将其连入pGPU6/GFP/Neo质粒中,构建AKT shRNA真核表达载体,经脂质体转染入Jeko-1细胞,应用RQ-PCR及Western blot鉴定其干扰效果,MTT法绘制细胞生长曲线,流式细胞术检测细胞凋亡的变化,Western blot检测凋亡相关蛋白信号及通路相关蛋白p-AKT、Notch1、HES1的变化。结果:AKT shRNA转染Jeko-1细胞后,AKT的mRNA和蛋白表达均明显下降,有统计学意义(P<0.05);转染组细胞增殖率明显低于Neg-shRNA组和空白对照组(P<0.05),AKT shRNA传染48 h后,凋亡率为(37.72±4.39)%,而NegshRNA组和空白对照组分别为(2.62±1.53)%、(1.57±0.42)%,差异有统计学意义(P<0.01);凋亡相关蛋白BCL-2表达下降,而BAX、caspase-3、caspase-9表达上升;PI3K/AKT信号通路相关蛋白p-AKT磷酸化水平下降、Notch1信号通路相关蛋白Notch1、HES1表达下调。结论:干扰沉默AKT基因可抑制套细胞淋巴瘤Jeko-1细胞增殖,诱导细胞凋亡,其机制可能是特异性抑制PI3K/AKT信号通路也能下调Notch1信号通路的活性。

Objective： To investigate the effects of silencing AKT gene by RNA interference on the proliferation, apoptosis and the expression of Notch1 signal pathway-related proteins in mantle cell lymphoma Jeko-1 cell line. Methods： The hairpin-like oligonucleotide sequences targeting AKT gene were designed and transfected into Jeko-1 cells by lipofectamineTM 2000. The AKT mRNA and protein were detected by RQ-PCR and Western blot respectively. Cell growth was determined by MTT. Cell apoptosis was analyzed by flow cytometry. The expression of BCL-2, BAX, caspase-3, caspase-9, p-AKT, Notch1, HES1 was detected by Western blot. Results： AKT mRNA was markedly suppressed by the shRNA targeting AKT. AKT shRNA suppressed the proliferation of Jeko-1 cells and induced apoptosis of these cells. The cell apoptotic rates were （ 37.72 ± 4.39 ） %, （ 2.62 ± 1.53 ） %, （ 1.57 ± 0.42 ） % in AKT shRNA, Neg-shRNA and Control, respectively, The difference between them was statistically significant （ P 〈 0.01 ）. AKT shRNA down-regulated the expression of Bcl-2 ,AKT p-AKT, Notchl, and HES1, up-regulated the expression of BAX, caspase-3 ,caspase-9. Conclusion： Silencing AKT gene can inhibit the proliferation of Jeko-1 cells line, induc cell apoptosis, its mechanism may be associated with specially inhibiting PI3K/AKT signaling pathway and down-regulating the activity of Notchl signaling pathway.