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. 2008 Mar;56(3):219-31.
doi: 10.1007/s10616-008-9151-2. Epub 2008 Jun 19.

Small interfering RNA knockdown of mini-TyrRS and mini-TrpRS effects angiogenesis in human umbilical vein endothelial cells in hypoxic culture

Rui Zeng  1 , Yu-Cheng Chen, Zhi Zeng, Rui Liu, Ou Qiang, Xiao-Fei Jiang, Xiao-Xia Liu, Xian Li, Hao-Yu Wang
Affiliations

Small interfering RNA knockdown of mini-TyrRS and mini-TrpRS effects angiogenesis in human umbilical vein endothelial cells in hypoxic culture

Rui Zeng et al. Cytotechnology. 2008 Mar.

Abstract

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.

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Figures

Fig. 1
Fig. 1
Schematic representation of human TyrRS, TrpRS and their variant constructs used in this study. Shaded regions of full-length TyrRS and TrpRS represent COOH- and NH2-terminal appended domains, respectively. Numbers on the left and right correspond to the NH2- and COOH-terminal residues relative to the human full-length enzymes, respectively
Fig. 2
Fig. 2
pGPU6/GFP/Neo expression vector containing a RNA polymerase III expression element. Silencing efficiency was assayed by real-time fluorescent quantitation PCR by expression of Green Fluorescence Protein (GFP)
Fig. 3
Fig. 3
Assay for mini-TrpRS gene silencing efficiency by Western blotting. Only the mini-TyrRS-specific siRNA inhibited the expression of mini-TyrRS protein. The silencing efficiency had no significant difference detected between the mock and untransfected groups. (1) Untransfected HUVECs (mock transfection); (2) negative control; (3) mini-TyrRS pGPU6/GFP/Neo expression vector transfected HUVECs (experimental group). Compared with GAPDH, protein expression of the full-length TyrRS maintained the same level from 3 to 24 h, but the mini-TyrRS protein expression increased during that time
Fig. 4
Fig. 4
Assay for mini-TyrRS gene silencing efficiency by Western blotting. Only the mini-TrpRS-specific siRNA inhibited the expression of mini-TrpRS protein. The silencing efficiency had no significant difference detected between the mock and untransfected groups. (1) Untransfected HUVECs (mock transfection); (2) negative control; (3) mini-TrpRS pGPU6/GFP/Neo expression vector transfected HUVECs (experimental group). Compared with GAPDH, protein expression of the full-length TrpRS maintained the same level from 3 to 24 h, but the Mini-TrpRS protein expression decreased during that time
Fig. 5
Fig. 5
Assay for mini-TyrRS/mini-TrpRS gene silencing efficiency by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR). Only the mini-TyrRS/mini-TrpRS specific siRNA inhibited expression of the mini-TyrRS/mini-TrpRS gene. No mini-TyrRS/mini-TrpRS knockdown was observed with the negative group. (1) Untransfected HUVECs (mock transfection); (2) negative control; (3) mini-TrpRS or mini-TyrRS pGPU6/GFP/Neo expression vector transfected HUVECs (experimental group)
Fig. 6
Fig. 6
Mini-TyrRS/mini-TrpRS-siRNA induced tube formation assay. *p < 0.05, compared with the mock transfection group and negative control group. There were no statistic difference between the mock transfection group and the negative control group
Fig. 7
Fig. 7
Assay of cell migration induced by mini-TyrRS/mini-TrpRS-siRNA. *p < 0.05, compared with the mock transfection group and negative control group. There were no statistic difference between the mock transfection group and the negative control group
Fig. 8
Fig. 8
Rate of cell proliferation induced by mini-TyrRS/mini-TrpRS-siRNA. *p < 0.05, compared with the mock transfection group and negative control group. There were no statistic difference between the mock transfection group and the negative control group
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