Study on Anti Epithelial-mesenchymal Transition (EMT)Effect by Knockout of Beta-catenin in Pulmonary Alveoli
|Keywords||β-catenin protein knockout TGF-β1 epithelial cells-mesenchymaltransition|
Objective:The study aims to investigate the effects of β-catenin knockout on TGF-β1induced epithelial-mesenchymal transition (EMT) in alveolar type Ⅱ epithelial cells which was transfected with β-catenin protein knockout vector (SCF～(β-TrCP)-Ecad). Furthermore, the present study explores the role of β-catenin signaling pathway in EMT induced by TGF-β1and the therapeutic effects of β-catenin protein knockout on EMT.Methods:The alveolar type Ⅱ epithelial cells (RLE-6TN) were cultured in-vitro and transiently transfected with empty plasmid (pcDNA3) and SCF～(β-TrCP)-Ecad, respectively followed by TGF-β1treatment (5ng/mL) for24h. Samples were divided into normal control group (group A), RLE-6TN plus TGF-β1group (group B), pcDNA3transfection plus TGF-β1group (group C) and SCF～(β-TrCP)-Ecad transfection plus TGF-β1group (group D). Protein levels of E-cadherin, α-smooth muscle actin (α--MA) and fibronectin (Fn) in each group were determined by western blot and mRNA levels of Snail, the downstream gene of β-catenin pathway, were detected by RT-PCR.Result:1. Western blot resulls of the expression of E-cadherin (E-cadherin), α-smooth muscle actin (α--MA) and fibronectin (Fn) of RLE-6TN cells treated with TGF-β1: Results showed that protein levels of α-SMA and Fn were significantly elevated, but expressions of E-cadherin were dramatically decreased in group B and C compared to group A (P<0.05). Differences between group B and C were not significant. Besides, expressions of α-SMA and Fn protein were significantly reduced, but E-cadherin expression was obviously elevated in group D compared with group B and C (P<0.05). No significant differences were found between group A and D.2. Real—time PCR resulls of the mRNA expression of β-catenin pathway downstream transcripts snail:Results showed that mRNA levels of Snail were significantly elevated in group B and C compared to group A (P<0.05). Differences between group B and C were not significant. Besides, expressions of levels of Snail were significantly reduced in group D compared with group B and C (P<0.05). No significant differences were found between group A and D.Conclusion:1. TGF-β1can induce EMT in RLE-6TN epithelial cells due to that cellular markers of mesenchyme were upregulated and markers of epithelial cells were downregulated. β-catenin is an important signalling molecule in the process of EMT induced by TGF-β1.2. Knockout of β-catenin protein reduced the cellular expression of β-catenin and blocked TGF-β1mediated EMT, leading to the inhibition of fibrosis process.