Fudan University, The Fifth People's Hospital of Shanghai, Department of Orthopedics - Shanghai, China.
Rev Assoc Med Bras (1992). 2021 Apr;67(4):555-560. doi: 10.1590/1806-9282.20201024.
In this study, we aimed at investigating the role of isoleucyl-tRNA synthetase in the growth, migration, and angiogenesis of human umbilical vein endothelial cells and the underlying molecular mechanism.
To assess the role of isoleucyl-tRNA synthetase, we silenced isoleucyl-tRNA synthetase in human umbilical vein endothelial cells using lentiviral 2 specific short hairpin RNAs (short hairpin RNAs 1 and 2) and examined silencing efficiency using real time quantitative polymerase chain reaction and western blot analyses. Short hairpin RNAs 1-isoleucyl-tRNA synthetase had greater knockdown efficiency, it was used in the entire downstream analysis. Short hairpin RNAs 1- isoleucyl-tRNA synthetase silencing effects on cell proliferation, cell colony generation, cell migration, as well as angiogenesis were assessed using cell counting kit-8, colony development, cell migration, and angiogenesis tube formation assays, respectively.
Compared to the control group, anti-isoleucyl-tRNA synthetase short hairpin RNAs significantly silenced isoleucyl-tRNA synthetase expression in human umbilical vein endothelial cells, and suppressed their proliferation, migration, and angiogenic capacity. To characterize the underlying mechanism, western blot analyses showed that isoleucyl-tRNA synthetase knockdown suppressed phosphorylation of extracellular-regulated kinase ½ and protein-serine- threonine kinase, as well as expression of vascular endothelial growth factor, GSK-3β, and β-catenin.
We have shown, for the first time, the critical role of isoleucyl-tRNA synthetase in human umbilical vein endothelial cells. Our data show that isoleucyl-tRNA synthetase knockdown suppresses human umbilical vein endothelial cell proliferation, migration, and angiogenesis. We have also shown that isoleucyl-tRNA synthetase knockdown suppresses phosphorylation of extracellular-regulated kinase ½ and protein-serine- threonine kinase, as well as expression of vascular endothelial growth factor, GSK-3β, and β-catenin. Together, these data highlight isoleucyl-tRNA synthetase as a potential antitumor anti-angiogenic target.
本研究旨在探讨亮氨酰 tRNA 合成酶在人脐静脉内皮细胞生长、迁移和血管生成中的作用及其潜在的分子机制。
为了评估亮氨酰 tRNA 合成酶的作用，我们使用慢病毒 2 种特异性短发夹 RNA（短发夹 RNA1 和 2）沉默人脐静脉内皮细胞中的亮氨酰 tRNA 合成酶，并使用实时定量聚合酶链反应和 Western blot 分析检测沉默效率。短发夹 RNA1-亮氨酰 tRNA 合成酶具有更高的敲低效率，因此在整个下游分析中都使用了它。使用细胞计数试剂盒-8、集落发育、细胞迁移和血管生成管形成测定法分别评估短发夹 RNA1-亮氨酰 tRNA 合成酶沉默对细胞增殖、细胞集落生成、细胞迁移和血管生成的影响。
与对照组相比，抗亮氨酰 tRNA 合成酶短发夹 RNA 显著沉默了人脐静脉内皮细胞中的亮氨酰 tRNA 合成酶表达，并抑制了它们的增殖、迁移和血管生成能力。为了表征潜在的机制，Western blot 分析表明，亮氨酰 tRNA 合成酶敲低抑制了细胞外调节激酶 1/2 和蛋白丝氨酸-苏氨酸激酶的磷酸化以及血管内皮生长因子、GSK-3β 和β-连环蛋白的表达。
我们首次表明亮氨酰 tRNA 合成酶在人脐静脉内皮细胞中具有关键作用。我们的数据表明，亮氨酰 tRNA 合成酶敲低抑制了人脐静脉内皮细胞的增殖、迁移和血管生成。我们还表明，亮氨酰 tRNA 合成酶敲低抑制了细胞外调节激酶 1/2 和蛋白丝氨酸-苏氨酸激酶的磷酸化以及血管内皮生长因子、GSK-3β 和β-连环蛋白的表达。总之，这些数据突出了亮氨酰 tRNA 合成酶作为潜在的抗肿瘤抗血管生成靶标。
