小干扰 RNA 敲低 mini-TyrRS 和 mini-TrpRS 对低氧培养人脐静脉内皮细胞血管生成的影响。
Small interfering RNA knockdown of mini-TyrRS and mini-TrpRS effects angiogenesis in human umbilical vein endothelial cells in hypoxic culture.
机构信息
Department of Cardiology, West China Hospital, School of Clinic Medicine, Sichuan University, Chengdu, 610041, China.
出版信息
Cytotechnology. 2008 Mar;56(3):219-31. doi: 10.1007/s10616-008-9151-2. Epub 2008 Jun 19.
Aim We studied the role of mini-TyrRS and mini-TrpRS in angiogenesis by using small interfering RNA-mediated mini-TyrRS/mini-TrpRS knockout in hypoxic culture of human umbilical vein endothelial cells. Methods SiRNA was used as the main method to inhibited the gene function. Silencing efficiency was assayed by real-time reverse transcription-polymerase chain reaction and western blotting. The angiogenic activity in vitro was evaluated by transwell migration assay and Matrigel-induced capillary tube formation in hypoxic culture. Cell proliferation was determined by crystal violet staining. Results The results showed that levels of the mini-TyrRS/mini-TrpRS gene and protein in mock transfection group and negative control group were higher, but noticeably decreased in experimental group. However, no significant difference was detected between mock transfection group and negative control group, but there was a statistically significant difference compared with experimental group. For mini-TyrRS-siRNA group, the cell migration, tube formation and the rate of cell proliferation were respectively inhibited by (47.4, 56.3, 65.4, 73.7%), (60.5, 69.1, 75.9, 83.6%) and (40.4, 56.2, 61.2, 68.0%). For mini-TrpRS-siRNA, were respectively increased by (18.0, 33.8, 45.1, 56.4%), (18.3, 31.2, 40.3, 45.7%) and (8.4, 26.4, 38.2, 46.6%). Conclusion These results indicated that angiogenesis is either stimulated by mini-TyrRS or inhibited by mini-TrpRS in matrigel models in hypoxic culture, raising the possibility that mini-TyrRS stimulates a common downstream signaling event. Thus, naturally occurring fragments of two proteins involved in translation, TyrRS and TrpRS, have opposing activity on endothelial cell angiogenesis in the matrigel assays. The opposing activities of the two tRNA synthetases suggest tight regulation of the balance between pro- and anti-angiogenic stimuli.
目的 我们通过在低氧培养的人脐静脉内皮细胞中使用小干扰 RNA 介导的 mini-TyrRS/mini-TrpRS 敲除来研究 mini-TyrRS 和 mini-TrpRS 在血管生成中的作用。
方法 采用小干扰 RNA 作为主要方法抑制基因功能。通过实时逆转录-聚合酶链反应和 Western blot 测定沉默效率。在低氧培养中通过 Transwell 迁移实验和 Matrigel 诱导的毛细血管形成评估体外血管生成活性。通过结晶紫染色测定细胞增殖。
结果 结果表明，mock 转染组和阴性对照组的 mini-TyrRS/mini-TrpRS 基因和蛋白水平较高，但实验组明显下降。然而，mock 转染组和阴性对照组之间无显著差异，但与实验组有统计学差异。对于 mini-TyrRS-siRNA 组，细胞迁移、管形成和细胞增殖率分别抑制了（47.4%、56.3%、65.4%、73.7%）、（60.5%、69.1%、75.9%、83.6%）和（40.4%、56.2%、61.2%、68.0%）。对于 mini-TrpRS-siRNA，分别增加了（18.0%、33.8%、45.1%、56.4%）、（18.3%、31.2%、40.3%、45.7%）和（8.4%、26.4%、38.2%、46.6%）。
结论 这些结果表明，在 Matrigel 模型中，低氧培养时血管生成受 mini-TyrRS 刺激或受 mini-TrpRS 抑制，提示 mini-TyrRS 刺激共同的下游信号事件。因此，参与翻译的两种蛋白质 TyrRS 和 TrpRS 的天然片段在 Matrigel 测定中对内皮细胞血管生成具有相反的活性。两种 tRNA 合成酶的相反活性表明，促血管生成和抗血管生成刺激之间的平衡受到严格调控。
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