eEF1A1回复表达对敲除eEF1A1基因的Jurkat细胞增殖、凋亡的影响及机制研究

Effects of eEF1A1 Re-expreesion on Proliferation and Apoptosis of Jurkat Cells with Knocked Down eEF1A1Gene and Its Mechanisms

本文探讨真核翻译延伸因子1A1(eEF1A1)回复表达对eEF1A1基因敲除人急性T淋巴细胞白血病(T-ALL)Jurkat细胞增殖、凋亡的影响及其作用机制。构建表达的eEF1A1慢病毒并感染eEF1A1基因敲除的Jurkat细胞,应用RT-PCR和Western blot分别检测细胞eEF1A1 mRNA和蛋白的表达;应用MTT法、Annexin V-APC标记法、DNA倍体法分别检测细胞增殖、细胞凋亡和细胞周期,Western blot法检测细胞PI3K/Akt信号通路相关信号分子的表达。结果表明,构建的eEF1A1表达慢病毒使基因敲除Jurkat细胞的eEF1A1表达明显回复。与阴性对照组相比,eEF1A1表达组Jurkat细胞增殖能力明显增强,凋亡明显减少,G0/G1期细胞比例明显减少;随着S期、G2/M期细胞比例增多,pAk、NF-κB、p-NF-κB、mTOR、p-mTOR蛋白的表达明显升高。结论:eEF1A1在T-ALL细胞中可能具有潜在的致癌作用,它的表达可有效促进Jurkat细胞的增殖并抑制凋亡,其机制可能与上调PI3K/Akt/NF-κB和PI3K/Akt/mTOR信号通路有关。

This study was aimed to explore the effects of expressing eukaryotic elongation factor 1A1 （eEF1A1） on proliferation and apoptosis in human acute T lymphocytic leukemia （T-ALL） cell line Jurkat with knocked down eEF1A1 gene and its mechanisms, eEF1Al-expressing lentivirus（LV） was constructed and used to transfect the Jurkat cells with knocked down eEF1A1 gene. Then, the expressions of eEF1A1 mRNA and protein were detected by real time PCR（RT- PCR） and Western blot respectively. Cell proliferation, apoptosis and cycle were detected by MTT method, Annexin V- APC labeling and DNA ploidy analysis respectively. The related protein expressions of phosphatidylinositol-3-kinase （P13K）/serine/threonine kinase （Akt） signaling pathway were detected by Western blot. The results indicated that eEF1A1 mRNA and protein expressions of Jurkat cells with knocked down eEF1A1 gene were re-established by constructing eEF1A1-expression LV. Compared with negative control group （transfected with negative control LV and eEF1A1-shRNA LV）, cell proliferation in eEF1A1 expression group was significantly enhanced, cell apoptosis was remarkably inhibited, percentage of cells in G0/G1 phase was significantly reduced alone With increased percentage of cells in S and G2/M phase, and the expression levels of p-Akt （Ser 473 ） , nuclear factor kappa B （NF-κB）, p-NF-κB （Ser 468）, mammalian target of rapamycin （roTOR） and p-mTOR （Set 2448 ） protein significantly increased. It is concluded that eEF1A1 may have a carcinogenic effect in T-ALL cells, eEF1A1 expression has noticeable effects on the proliferation enchancement and apoptosis inhibition of Jurkat cells, which may be mediated by the up-regulation of P13K/ AM/NF-KB and P13K/Akt/mTOR signaling pathway.