Wnt3a-induced extracellular matrix production and gel contraction in human lens epithelial cells

Authors:Bao Xiuli,  Liu Tingting,  Wang Yanyan,  Li Lin
DOI: 10.3760/cma.j.issn.2095-0160.2016.07.005
Published 2016-07-10
Cite as Chin J Exp Ophthalmol, 2016,34(7): 597-601.

Abstract

Background

Excessive production, deposition and contraction of extracellular matrix (ECM) of human lens endothelial cells (LECs) is one of main causes to posterior capsular opacification (PCO). Researches indicated that Wnt3a protein was involved in production of ECM and fibrosis in epithelial cells, but its effect on LECs is still unclear.

Objective

This study aimed to elucidate the roles of Wnt3a in production of ECM and gel contraction of LECs.

Methods

Lipofectamine-mediated transient transfection technique was used to introduce cDNA of Wnt3a gene and pcDNA3-HA vector into the LEC cell line SRA01/04 to act as Wnt3a transfection group and control group respectively.After 48 h of transfection, Western blot was used to detect the expression of Wnt3a, main components of ECM Col-Ⅰ, Col-Ⅳ and integrin β1.Immunofluorescence was used to detect the expression and distrubition of α-SMA and F-actin; collagen contraction was observed by mingling SRA01/04 cells with Col-Ⅰ.

Results

After 48 hours of transfection using lipofectamine 2000, the expressions of wnt3a protein in SRA01/04 cells was 0.703±0.105 in the wnt3a transfected group, and that in the control group was 0.290±0.066, showing a significant difference between the two groups (t=5.782, P<0.01). Western blot assay showed that the expression levels of Col-Ⅰ and Integrin β1 was 0.697±0.021 and 0.875±0.055 in the Wnt3a transfected group, and which was significantly higher than 0.370±0.020 and 0.580±0.030 in the control group (t=19.600, 8.156, both P<0.01). The expression level of Col Ⅳ in the Wnt3a transfected group was higher than that of the control group (0.430±0.020 vs 0.383±0.031), but the difference was not significant (t=2.514, P>0.05). Immunofluorescence assay revealed that F-actin and α-SMA were weakly expressed in the cell membrane primarily in the control group, while they were strongly expressed in the cell membrane, cytoplasma in the Wnt3a transfected group.Tewnty-four hours after addtion of Col-Ⅰ, the gel contraction area ratio appeared to be more obvious in comparison with the 8 hours (64.1%±2.3% vs 98.9%±1.0%), and gel contraction area ratio was lower in the Wnt3a transfected group than that in the control group (64.1%±2.3% vs 93.9%±3.1%).

Conclusions

The overexpression of Wnt3a activates the production of ECM, and the remodeling of celluar skeleton and cellular contraction.

Key words:

Epithelial-mesenchymal transition; Epithelial cells, lens; Lens capsule, crystalline/cytology; Extracellular matrix; Posterior capsular opacification; Wnt signaling pathway; Transfection

Contributor Information

Bao Xiuli
Department of Ophthalmology, Affiliated Hospital of Inner Mongolia Medical University, Huhhot 010050, China
Liu Tingting
Wang Yanyan
Li Lin
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Updated: October 8, 2019 — 9:23 am