eEF1A1基因敲除对Jurkat细胞增殖和凋亡的影响及其机制探讨

Effect of Knocking down eEF1A1 Gene on Proliferation and Apoptosis in Jurkat Cells and Its Mechanisms

本研究旨在探讨真核翻译延长因子1A1(eEF1A1)表达沉默对人急性T淋巴细胞白血病(T-ALL)细胞株Jurkat增殖、凋亡的影响及其作用机制。应用实时PCR法和Western blot法分别检测Jurkat细胞和3例健康成人外周血单个核细胞(PBMNC)中eEF1A1 mRNA和蛋白的表达。构建eEF1A1-shRNA慢病毒并感染Jurkat细胞,另设空白和阴性对照组,应用实时PCR法和Western blot法分别检测细胞eEF1A1 mRNA和蛋白的表达;应用MTT法、AnnexinⅤ-APC标记法、DNA倍体法分别检测细胞增殖、细胞凋亡、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,Jurkat细胞eEF1A1 mRNA和蛋白的表达水平明显高于健康成人PBMNC中的表达(P<0.01,P<0.05);构建的eEF1A1-shRNA慢病毒高效沉默Jurkat细胞eEF1A1的表达。与阴性对照组相比,eEF1A1-shRNA组Jurkat细胞增殖能力明显下降,凋亡明显增多,细胞周期被阻滞于G0/G1期,p-Akt、NF-κB、p-NF-κB、mTOR、p-mTOR蛋白的表达明显下调。结论:eEF1A1在T-ALL细胞中可能具有潜在的致癌作用,其表达沉默可有效抑制Jurkat细胞的增殖并诱导凋亡,其机制可能与下调PI3K/Akt/NF-κB和PI3K/Akt/mTOR信号通路有关。

This study was purposed to investigate the effect of knocking down eukaryotic elongation factor 1A1 （ eEF1A1 ） gene on the proliferation and apoptosis in human acute T lymphocytic leukemia （T-ALL） cell line Jurkat and explore its mechanism. The eEF1A1 mRNA and protein expressions of Jurkat cells and 3 healthy adult peripheral blood mononuclear cells （PBMNC） were detected by real time PCR and Western blot, respectively, eEF1A1-shRNA lentivims was constructed through molecular biological method, and was used to transfect Jurkat cells. Then, cell eEF1A1 mRNA and protein expressions were detected by real time PCR and Western blot, respectively. Cell proliferation, apoptosis and cycle were detected by MTT method, Annexin V-APC labeling and DNA ploidy analysis, respectively. Cell-related protein expressions of phosphatidylinositol-3-kinase （ PI3K）/serine/threonine kinase （Akt） signaling pathway were detected by Western blot. The results showed that eEF1A1 mRNA and protein expression levels of Jurkat cells were significantly higher than that of healthy adult PBMNC, respectively （P 〈 0.01, P 〈 0.05 ）. eEF1A1 mRNA and protein expressions of Jurkat cells were significantly knocked down by constructing eEF1A1-shRNA lentivirus. Compared to negative control group （transfected with negative control-shRNA lentivirus） ,cell proliferation in eEF1A1-shRNA group was significantly inhibited, cell apoptosis was remarkably induced, cell cycle was blocked in G0/G1 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 （mTOR） and p-mTOR （ Ser 2448） proteins were significantly reduced. It is concluded that eEF1A1 may be a putative oncoprotein in T-ALL cells. Knocking down eEF1A1 gene has noticeable effects on the proliferation inhibition and apoptosis induction of Jurkat cells, which may be mediated by the down-regulation of PI3K/Akt/NF-κB and PI3K/ Akt/ mTOR signaling pathway.