Abstract
Aim:
To investigate the effect of lentivirus-mediated integrin-linked kinase (ILK) RNA interference (RNAi) on human retinal Müller cells transdifferentiation into contractile myofibroblasts.
Methods:
A lentiviral vector expressing ILK-specific shRNA was constructed and introduced into cultured retinal Müller cells. Silencing of the ILK gene was identified by real time RT-PCR and Western blot. The Müller cell phenotype change was confirmed by immunodetection of α-smooth muscle actin (α-SMA) stress fiber formation. The generation of tractional force was assessed using a tissue culture assay with cells incubated in three-dimensional collagen gels; cell migration was determined by the Boyden chamber method, using 10% FBS as a chemotactic factor.
Results:
Significant decreases in ILK mRNA and protein expression were detected in Müller cells carrying lentiviral ILK-shRNA vector. Cells treated with anti-ILK siRNA showed less α-SMA stress fiber formation under hypoxic conditions or cell subcultivation. Lentiviral ILK-shRNA vector transfection also significantly reduced cell migration and cell-mediated gel contraction.
Conclusion:
Lentivirus-mediated ILK RNAi decreased cell migration and contractile force generation by inhibiting α-SMA stress fiber formation in human retinal Müller cells. This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.
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This work was funded by the National Natural Science Foundation of China (No 30772598).
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Zheng, Yp., Liu, H., Zeng, H. et al. Downregulation of lentivirus-mediated ILK RNAi on tractional force generation in human retinal Müller cells. Acta Pharmacol Sin 30, 1625–1633 (2009). https://doi.org/10.1038/aps.2009.154
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DOI: https://doi.org/10.1038/aps.2009.154
