Abstract
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.
Highlights
Angiogenesis, the physiological process through which new blood vessels are formed, provides oxygen and nutrients to actively proliferating tumor cells[1]
To assess the role of IARS2, we knocked down IARS2 in HUVEC cells using two specific shRNAs and examined IARS2 titers by western blot analysis, as well as RT-qPCR assays (Figure 1)
CCK-8 analysis revealed that IARS2-silenced HUVECs grew slower than control cells (Figure 2A)
Summary
Angiogenesis, the physiological process through which new blood vessels are formed, provides oxygen and nutrients to actively proliferating tumor cells[1]. To support the high proliferative degree of cancer cells, tumors should rapidly form new vascular networks[2]. Multiple clinical studies show that tumors are angiogenesis dependent. This process is needed to support the growth of tumors beyond 1–2 mm[3]. Anti-angiogenesis has arisen as a prospective anti-cancer approach[3]. Angiogenesis is strongly modulated by an equilibrium between pro- and anti-angiogenic factors. Multiple factors like VEGF, neuropilin-1, tyrosine kinase with Ig, and bFGF are involved in angiogenesis. Elevated tumor VEGF levels are a key feature in tumor angiogenesis[4].
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