Knockdown of Multidrug Resistance-Associated Protein 4 by Sirna Enhances Cell Migration and Tube Formation in Human Retinal Endothelial Cells

Abstract

Purpose: : To explore the angiogenic function of multidrug resistance-associated protein 4 (Mrp4), an efflux ATP-binding cassette transporter , in human retinal endothelial cell (HREC) culture.

Methods: : Mrp4 gene and protein expressions in HRECs were examined by RT-PCR analysis and immunohistochemical analysis, respectively. Semiconfluent HREC cultures were transfected with Mrp4 siRNA or nonsilencing siRNA and used in the following assays 24 hours after transfection. HREC proliferation was evaluated using DNA synthesis measurements by 5-bromo-2’-deoxyuridine (BrdU) incorporation and HREC migration was assessed with a modified Boyden chamber assay. The effect of Mrp4 knockdown on the ability of capillary-like formation was tested by tube formation assay in Matrigel.

Results: : HRECs expressed Mrp4 mRNA and showed immunoreactivity for Mrp4. Knockdown of Mrp4 by siRNA enhanced the migratory capacity of HRECs (P = 0.002). Similarly, Mrp4-knockdown HRECs showed a marked increase in tube formation (P = 0.001). On the other hand, there was no significant difference in cell proliferation between Mrp4-knockdown and control HRECs (P = 0.134).

Conclusions: : Transfection of Mrp4 siRNA promotes cell migration and tube formation in HRECs. These results suggest that Mrp4 is negatively associated with retinal angiogenesis.

Keywords: retinal neovascularization • cell membrane/membrane specializations • retinal culture