Abstract
Purpose :
Fibroblast growth factor (FGF) signaling regulates posterior capsular opacity, (PCO) cell proliferation and lens fiber cell differentiation after cataract surgery. We previously reported that the Tropomyosin (Tpm) family of cytoskeleton proteins is induced by TGFβ2 during epithelial–mesenchymal transition (EMT) and repressed by FGF2 in lens epithelial cells (LECs) and is related to the progression of PCO. We also reported that miR29c is related to the lens development and Tpm is one of target gene of miR29c. In this study, we investigated the involvement of microRNA 29c (miR29c) in repression of Tpm1 and 2 expressions by FGF2.
Methods :
Primary cultured mouse LECs (MLEC) growing in DMEM containing 2% fetal bovine serum were treated with 0 to 10ng/ml of FGF2. To identify micro RNA expression in MLEC, real-time PCR was used to analyze expressions of miR29c and Tpm1/2. For inhibition of miR29c, miRIDIAN Hairpin inhibitor, mouse miR 29c were used and transfected into MLEC. For overexpression of miR29c, miRNA precursors were transfected into MLEC. Data were from three experiments and ere reported as means ± SDs and analyzed by 1-way ANOVA, followed by a t test when appropriate, with p< 0.05 deemed significant.
Results :
MLECs treated with FGF2 for 2- and 4-days showed a significant induction in the expressions of miR29c (Fold change: 2.4 ± 0.42 and 2.48 ± 0.68, respectively; p<0.02) compared to negative control and a reduction in the expression of Tpm1/2 (p<0.001). However, in MLECs transfected by miR29c inhibitors, expression of Tpm1/2 was not reduced after addition of FGF2. Transfection experiments by mouse miRNA precursor revealed that the increasing level of miR29c (p<0.04) caused a corresponding decrease in expression of Tpm1/2, suggesting the involvement of miR29c in the translational regulation of Tpm1 and 2 by FGF2. Furthermore, 3’UTR luciferase activity of Tpm1, not Tpm2, was significantly decreased in miR29c transfected MLEC (p<0.02). From these results, we hypothesized that Tpm1 expression may be directly regulated by miR29c. Further, Tpm2 expression may be indirectly regulated by miR29c.
Conclusions :
These findings demonstrate that FGF2 may suppress Tpm1/2 via the upregulation of miR29c expression. miR29c may contribute to progression of PCO and lens fiber differentiation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.