Downregulation of lentivirus-mediated ILK RNAi on tractional force generation in human retinal Müller cells.
OBJECTIVE
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 alpha-smooth muscle actin (alpha-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 alpha-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.


CONCLUSIONS
Lentivirus-mediated ILK RNAi decreased cell migration and contractile force generation by inhibiting alpha-SMA stress fiber formation in human retinal Müller cells. This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.
Zheng
Yu-ping
YP
Department of Ophthalmology, 2nd Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, China.
Liu
Hui
H
Zeng
Hao
H
Xiong
Lei
L
Feng
Zhao-hui
ZH
Sun
Nai-xue
NX
D000199:Actins / Q000378:metabolism; D002454:Cell Differentiation; D002465:Cell Movement; D002478:Cells, Cultured; D015536:Down-Regulation; D005347:Fibroblasts / Q000473:pathology / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus; D009130:Muscle, Smooth / Q000378:metabolism; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012160:Retina / Q000166:cytology*; D022502:Stress Fibers / Q000378:metabolism*; D000099200:Scaffold Protein ILK
D000199:Actins; D017346:Protein Serine-Threonine Kinases; D000099200:Scaffold Protein ILK