Targeting of integrin-linked kinase with a small interfering RNA inhibits endothelial cell migration, proliferation and tube formation in vitro

Lili Guo, Wenzhen Yu, Xiaoxin Li, Gang Zhao, Peiying He
Ophthalmic Research 2009, 42 (4): 213-20

PURPOSE: To investigate the role of integrin-linked kinase (ILK) in endothelial cell migration, proliferation and tube formation in vitro.

MATERIALS AND METHODS: Cultured RF/6A cells were knocked down for ILK using small interfering RNA (siRNA). Cellular ILK expression was quantified by real-time quantitative PCR and Western blot analysis. Cell migration was measured by cell counting in modified Boyden chambers, while microfilament dynamics were assessed by immunofluorescence analysis. Cell cycling was determined using a FACS Calibur flow cytometer, and the expression of cyclin D(1) was also measured by the Western blot assay. The secretion of vascular endothelial growth factor (VEGF) in culture medium samples was assessed by ELISA, and capillary/tube-like network formation assays were performed using matrigel.

RESULTS: Both ILK mRNA and protein levels were virtually undetectable after transfection with ILK siRNA. In addition, there was a significant accumulation of cells in the G(0)-G(1) phase and a marked decrease in cell numbers in the S phase in ILK-specific siRNA-transfected cells, and the expression of cyclin D(1) was decreased after transfection. The knockdown of ILK significantly inhibited cell migration ability by disturbing F actin assembly, and VEGF secretion in conditioned medium was also reduced by 33%. Furthermore, treatment with ILK siRNA suppressed tube formation in RF/6A cells and significantly reduced the overall length of the tubes.

CONCLUSIONS: Knockdown of ILK with siRNA effectively inhibits endothelial cell migration, proliferation and tube formation in vitro.

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