VE-statin/Egfl7 siRNA inhibits angiogenesis in malignant glioma in vitro

Abstract

This study investigated the role of VE-statin/Egfl7 and its mechanism in angiogenesis in malignant glioma. Transwell culture plates were used to establish an U251-HUVEC co-culture system, which was used to mimic the interaction between malignant glioma and endothelial cells. Lentiviral vectors expressing VE-statin/Egfl7 siRNA were constructed, and U251 cells and HUVECs were transfected to inhibit VE-statin/Egfl7 expression. The proliferation, adherence, migration, and lumen formation of endothelial cells were assayed to investigate the influence of VE-statin/Egfl7 on angiogenesis in malignant glioma in vitro. Data showed that HUVEC growth was temporarily slowed after silencing the VE-statin/Egfl7 gene but rapidly returned to normal. Although endothelial cell migration was not influenced, cell adherence was markedly inhibited. Furthermore, the endothelial cells failed to generate a capillary-like lumen after VE-statin/Egfl7 gene silencing. Therefore, it can be concluded that VE-statin/Egfl7 may regulate the adherence of endothelial cells, thus playing an important role in endothelium-induced lumen formation during angiogenesis in malignant glioma.

Keywords: Epidermal growth factor-like domain 7; RNA interference; angiogenesis; malignant glioma.

Figure 1

Fluorescence detection of HUVEC and U251 cells infected with the lentiviral expression vectors of siRNA targeting VE-statin/Egfl7. (A-C) Represented normal negative control HUVECs (A), the HUVECs infected with universal negative control lentiviral (B) or VE-statin/Egfl7-siRNA lentivirus (C) respectively; (D-F) Represented normal negative control U251 (D), the U251 infected with universal negative control lentiviral (E) or VE-statin/Egfl7-siRNA lentivirus (F) respectively.

Figure 2

Effect of VE-statin/Egfl7 siRNA on the proliferation of HUVEC cells. (A, B) HUVECs proliferation at 48 h after incubation in group KD was significantly lower than that in the other two group, and this status continued to 4 d after incubation detected by cell proliferation assay (*, # vs. KD group, q=20.023, 16.686; 29.009, 26.034; 30.549, 28.672, P<0.01). Represented normal negative control HUVECs (A), the HUVECs infected with universal negative control lentiviral (B) or VE-statin/Egfl7-siRNA lentivirus (C) respectively

Figure 3

Effect of VE-statin/Egfl7 siRNA on the adhesion and tube formation of HUVEC cells. (A-C) Endothelial cell adhesion assay represented normal negative control HUVECs (A), the HUVECs infected with universal negative control lentiviral (B) or VE-statin/Egfl7-siRNA lentivirus (C) respectively; (D-F) Tube formation assay represented normal negative control HUVECs (D), the HUVECs infected with universal negative control lentiviral (E) or VE-statin/Egfl7-siRNA lentivirus (F) respectively.