Huang Yi, Hu Jian-Da, Wu Yan-An, Zheng Jing, Qi Yuan-Lin, Chen Ying-Yu, Huang Xiao-Li
Fujian Medical University, Fuzhou, Fujian Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Apr;21(2):279-84. doi: 10.7534/j.issn.1009-2137.2013.02.004.
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. eEF1A1-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 (PI3K)/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 (mTOR) and p-mTOR (Ser 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 enhancement and apoptosis inhibition of Jurkat cells, which may be mediated by the up-regulation of PI3K/Akt/NF-κB and PI3K/Akt/ mTOR signaling pathway.
本研究旨在探讨真核生物延伸因子1A1（eEF1A1）过表达对eEF1A1基因敲低的人急性T淋巴细胞白血病（T-ALL）细胞系Jurkat增殖和凋亡的影响及其机制。构建了表达eEF1A1的慢病毒（LV），并用于转染eEF1A1基因敲低的Jurkat细胞。然后，分别通过实时荧光定量聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测eEF1A1 mRNA和蛋白质的表达。分别采用MTT法、膜联蛋白V-别藻蓝蛋白（Annexin V-APC）标记法和DNA倍体分析检测细胞增殖、凋亡和周期。通过蛋白质免疫印迹法检测磷脂酰肌醇-3激酶（PI3K）/丝氨酸/苏氨酸激酶（Akt）信号通路的相关蛋白表达。结果表明，通过构建表达eEF1A1的LV，可使eEF1A1基因敲低的Jurkat细胞重新建立eEF1A1 mRNA和蛋白质表达。与阴性对照组（转染阴性对照LV和eEF1A1-shRNA LV）相比，eEF1A1表达组细胞增殖显著增强，细胞凋亡明显受到抑制，G0/G1期细胞百分比显著降低，S期和G2/M期细胞百分比增加，且p-Akt（Ser 473）、核因子κB（NF-κB）、p-NF-κB（Ser 468）、雷帕霉素靶蛋白（mTOR）和p-mTOR（Ser 2448）蛋白表达水平显著升高。结论：eEF1A1在T-ALL细胞中可能具有致癌作用。eEF1A1表达对Jurkat细胞增殖增强和凋亡抑制具有显著影响，其机制可能是通过上调PI3K/Akt/NF-κB和PI3K/Akt/mTOR信号通路介导的。
