Fibronectin and focal adhesion kinase small interfering RNA modulate rat retinal Müller cells adhesion and migration

Abstract

Retinal Müller cells (RMCs) hypertrophy and proliferation play a crucial role in epiretinal membrane formation. This study was designed to analyze the effects of Fibronectin and specific FAK siRNA in cell adhesion and migration in rat Müller cells. RMCs were cultured and identified by GFAP, Vimentin, and GLAST mAb, respectively. The cells were planted on dishes coated with Fibronectin at 0, 1, 5, 10, 50, and 100 microg/ml. The attachment and migration assay was applied to characterize the RMCs-Fibronectin interactions. Cell lysis and Western blotting were utilized to detect beta(1)-integrin, FAK, and GLAST protein expression. Then the cells were treated with FAK siRNA, non-targeting siRNA, and control medium. The cell cycle and apoptosis rate was determined by flow cytometry. The attachment, migration, and Western blotting assay were repeated. These data suggested that almost all the cells expressed GFAP, Vimentin, and GLAST, respectively, which ensured most of the harvested cells were RMCs. In attachment assay, the A570 values increased significantly with time (F = 1105.439, P < 0.001) and Fibronectin concentration (F = 424.683, P < 0.001). There were significant difference between each Fibronectin concentration in RMCs migration (F = 34.703, P < 0.000). The expression ratio of FAK, beta(1)-integrin, and GLAST elevated significantly as Fibronectin concentration increased (F = 54.755, P < 0.000; F = 119.962, P < 0.000; F = 39.287, P < 0.000). The Fibronectin pretreatment was settled on 50 microg/ml for siRNA inhibition assays. The specific FAK siRNA treatment significantly increased G(0)/G(1) percentage and apoptosis rate compared with NT siRNA and control group (F = 11.526, P = 0.009; F = 64.772, P < 0.000). The apoptotic rate was significantly suppressed by inhibitors of caspase-8 and 3 (F = 10.500, P = 0.011). The A570 values were significantly suppressed in FAK siRNA groups compared with NT siRNA and control group (F = 154.241, P < 0.000), and the mean migratory cells per view field were significantly decreased (F = 10.906, P = 0.001). FAK and GLAST expression ratio decreased significantly after FAK siRNA treatment (F = 5.315, P = 0.047; F = 5.985, P = 0.042). Take together, FAK is involved in beta(1)-integrin mediated adhesive signaling and play a critical role in regulating Müller cell adhesion, migration, and so far as to glutamate transportation functions.

Fig. 1

Images of RMCs identification is in this part. a The cell culture image illustrated the retinal glial cell. b RMCs can express GFAP in cytoplasm and axons following activation or retinal damage. c Vimentin is exclusively found in RMCs. d GLAST proteins were characterized as discrete dot-like patterns predominantly distribute in the membrane, cytoplasm, and axons of RMCs

Fig. 2

Various concentrations of Fibronectin promote RMCs attachment and migration. a The A570 values increased gradually with time and Fibronectin concentration. b The mean migratory cells per view field reached its peak point with 50 μg/ml Fibronectin, and decreased in 100 μg/ml treatment. The optic Fibronectin concentration for RMCs attachment and migration was chosen as 50 μg/ml

Fig. 3

The RMCs attachment and migration were inhibited after FAK siRNA treatment. a The A570 values decreased significantly after siRNA treatment compared with the NT siRNA and the control groups. The inhibition rates were 20.13%, 18.21%, 25.46%, 30.79%, and 31.37% at each time point, respectively. b The mean migratory cells per view field decreased in FAK siRNA group. The inhibition rate was 25.77%. And the FAK siRNA induced migration inhibition mainly resulted from the decrease in cell adhesion