Study On Wnt Signaling Inducing Epithelial Mesenchymal Transition And Proliferation Of Human Lens Epithelial Cells

ObjectivesPosterior capsular opacification (PCO) is the most common posteoperative complication of modern cataract surgery causing visual loss. Epithelial mesenchymal transition(EMT) and proliferation of lens epithelial cells play important role in development of PCO. Wnt ligands is involved in regulation in proliferation, migration and transdifferentiation of diverse cells. However, the role of wnt3a ligand inducing canonical wnt signaling in lens epithelial cells remains unclear. In our study, we constructed wnt3a expression vector activating canonical wnt signaling pathway and used transfection technology to achieve wnt3a-overexpressing lens epithelial cells, investigated the influence of canonical wnt signaling pathway in EMT and proliferation of lens epithelial cells and its related mechanism, and try to find new target to design drugs.Methods1. We constructed Wnt3a overexpressing vector, then transformed into E.coli, confirmed by restrict enzyme digestion and sequence analysis.2. Using Lipo2000transfection technology, the Wnt3a vector was tranfected into human lens epithelial cells HLE B-3cells and SRA01/04cells. The level of Wnt3a protein was detected by westen blotting technique.3. Transwell assay and wound healing assay were performed to detect the role of Wnt3a in cell movement.4. Using MTT and flow cytometery analysis to detect the influence of Wnt3a overexpression on HLE B-3cells and SRA01/04cells proliferation.5. Using western blotting technique to detect β-catenin activation induced by Wnt3a overexpression, immunofluorescence assay to detect β-catenin location and intracellular distribution, for identify the state of Wnt/β-catenin signaling pathway.6. Using western blotting technique to detect the changes of EMT protein markers and nuclear target genes, and immunoflueorescence assay to detect the locations of those proteins and genes, for further investigate the mechanisms of Wnt/β-catenin in development of PCO.Results1. The Wnt3a-pcDNA3recombinated vector was constructed successfully, the sequence analysis was identical with Genebank.2. The expression of Wnt3a was less in normal lens epithelial cells. The lens epithelial cells with overexpressing Wnt3a was achieved by transient transfection with Wnt3a-pcDNA3vector.3. After tranfection with Wnt3a vector, the expression of β-catenin was increased in HLE B-3cells and slightly increased in SRA01/04cells, and the cytoplasmic β-catenin was translocatied into nucellus in HLE B-3cells and SRA01/04cells.4. Wnt3a-overexpressed HLE B-3cells and SRA01/04cells achieved ability of migration and motility compared to control cells(P<0.01).5. Wnt3a improved the proliferation of HLE B-3cells and SRA01/04cells(P<0.01), cell cycle analysis showed that number of cells stopping in S phase was increased in wnt3a-overexpressed cells compared with control cells.6. Compared with control cells, the E-cadherin as epithelial biomarker was down-regulated and fibronection as mesenchymal biomarker was upregulated in wnt3a-overexpressed HLE B-3cells and SRA01/04cells; and the immunofluoresecence analysis further identified the changes of EMT in wnt3a-overexpressed HLE B-3cells and SRA01/04cells.7. Wnt3a induced the up-regulation of Wnt/β-catenin nuclear target genes including Cyclin D1and c-Myc proteins in wnt3a-overexpressed HLE B-3cells and SRA01/04cells.Conelusions1. Wnt3a-pcDNA3recombinated vector efficatiously up-regulated the expression of Wnt3a protein, then activated the Wnt/β-catenin signaling pathway in human lens epithelial cells.2. Wnt3a improved the activity of motility and migration in human lens epithelial cells.3. Cyclin D1and c-Myc are downstream signaling molecules of Wnt/p-catenin signaling pathway. Wnt3a improved proliferation in human lens epithelial cells by activating Wnt/β-catenin signaling pathway and upregulating the downstream target proteins including Cyclin D1and c-Myc.4. Wnt3a induced EMT in lens epithelial cells.