eEF1A2 knockdown impairs neuronal proliferation and inhibits neurite outgrowth of differentiating neurons
eEF1A2 knockdown impairs neuronal proliferation and inhibits neurite outgrowth of differentiating neurons
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eEF1A2 敲低损害神经元增殖并抑制分化神经元的神经突生长
作者：
Kanitin Rumpansuwon;Athinan Prommahom;P. Dharmasaroja
目的 翻译延伸因子 1, α-2 (eEF1A2) 在蛋白质合成中发挥重要作用。已经在患有神经发育障碍的个体中描述了该基因的突变。在这里，我们沉默了人 SH-SY5Y 神经母细胞瘤细胞中 eEFA2 的表达，并观察了其在视黄酸诱导后在神经元增殖和分化中的作用。方法使用 siRNA 转染沉默 eEF1A2。通过 Ki-67 免疫细胞化学定性评估细胞增殖。用视黄酸诱导神经元分化3、5、7和10天。测量神经突长度。通过蛋白质印迹分析微管相关蛋白 2 (MAP2) 的表达。通过免疫荧光可视化酪氨酸羟化酶表达。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴化物 (MTT) 测定和裂解 caspase-3 的蛋白质印迹法评估神经毒素 1-甲基-4-苯基吡啶鎓 (MPP+) 的细胞毒性。结果 eEF1A2 敲低抑制了未分化 SH-SY5Y 细胞的增殖活性，如 Ki-67 免疫染色减少所示。在视黄酸诱导后，eEF1A2敲低的分化神经元表现出比未转染细胞更短的神经突长度，这与分化10天时酪氨酸羟化酶的减少和MAP2的抑制有关。 eEF1A2 敲低降低了神经元的存活率，这在未分化和短期分化细胞中可以清楚地观察到。经过 MPP+ 处理后，eEF1A2 敲低的细胞显示出细胞存活率进一步降低，并且裂解的 caspase-3 蛋白增加。结论 我们的结果表明 eEF1A2 可能是 SH-SY5Y 细胞神经元增殖和分化所必需的。 eEF1A2 敲低神经元中对 MPP+ 的细胞死亡敏感性增加可能意味着 eEF1A2 的神经保护作用。
Objectives The translation elongation factor-1, alpha-2 (eEF1A2) plays an important role in protein synthesis. Mutations in this gene have been described in individuals with neurodevelopmental disorders. Here, we silenced the expression of eEFA2 in human SH-SY5Y neuroblastoma cells and observed its roles in neuronal proliferation and differentiation upon induction with retinoic acid. Methods eEF1A2 were silenced using siRNA transfection. Cell proliferation was qualitatively evaluated by Ki-67 immunocytochemistry. Neuronal differentiation was induced with retinoic acid for 3, 5, 7 and 10 days. Neurite length was measured. The expression of microtubule-associated protein 2 (MAP2) was analyzed by western blotting. Tyrosine hydroxylase expression was visualized by immunofluorescence. Cytotoxicity to a neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and western blotting of cleaved caspase-3. Results eEF1A2 knockdown suppressed the proliferative activity of undifferentiated SH-SY5Y cells as shown by decreased Ki-67 immunostaining. Upon retinoic acid-induction, differentiated neurons with eEF1A2 knockdown exhibited shorter neurite length than untransfected cells, which was associated with the reduction of tyrosine hydroxylase and suppression of MAP2 at 10 days of differentiation. eEF1A2 knockdown decreased the survival of neurons, which was clearly observed in undifferentiated and short-term differentiated cells. Upon treatment with MPP+, cells with eEF1A2 knockdown showed a further reduction in cell survival and an increase of cleaved caspase-3 protein. Conclusions Our results suggest that eEF1A2 may be required for neuronal proliferation and differentiation of SH-SY5Y cells. Increased cell death susceptibility against MPP+ in eEF1A2-knockdown neurons may imply the neuroprotective role of eEF1A2.