Effects of β-catenin on the proliferative activity of and expressions of two apoptosis-related genes Bcl-2 and Bax by human skin fibroblasts induced by hydrogen peroxide

Abstract

Objective To investigate the effect of highly expressed β-catenin on the proliferative activity of and expressions of two apoptosis-related genes Bcl-2 and Bax by human skin fibroblasts induced by hydrogen peroxide（H2O2）. Methods Normal human skin fibroblasts（HSFs）from child foreskin were divided into three groups: empty vector group transfected with the empty vector pcDNA3.1, H2O2 group transfected with the empty vector pcDNA3.1 followed by treatment with H2O2（150 μmol/L）for 2 hours, β-catenin group transfected with pcDNA3.1-β-catenin followed by treatment with H2O2（150 μmol/L）for 2 hours. Subsequently, methyl thiazolyl tetrazolium（MTT）assay was conducted to estimate proliferative activity of fibroblasts, flow cytometry to detect cell apoptosis, and reverse transcription（RT）-PCR and Western blot were performed to measure the mRNA and protein expressions of Bcl-2 and Bax respectively. The relative expression levels of genes were expressed as the ratios between the targets and GAPDH. Results Significant differences were found between the empty vector group, H2O2 group and β-catenin group in cellular proliferative activity（expressed as absorbance value at 570 nm: 0.792 ± 0.012 vs. 0.462 ± 0.012 vs. 0.521 ± 0.015, P < 0.01）and apoptosis rate（3.407% ± 0.217% vs. 24.555% ± 1.793% vs. 15.360% ± 0.755%, P < 0.01）. Both mRNA and protein expression levels of Bcl-2 were significantly lower in the H2O2 group（0.333 ± 0.003 and 0.336 ± 0.004 respectively）than in the empty vector group（0.507 ± 0.013 and 0.514 ± 0.021, respectively, both P< 0.01）and β-catenin group（0.404 ± 0.006 and 0.411 ± 0.005, respectively, both P < 0.01）. Increased expression levels of Bax mRNA and protein were observed in the H2O2 group compared with the empty vector group and β-catenin group（mRNA: 0.451 ± 0.002 vs. 0.303 ± 0.005 and 0.339 ± 0.012, protein: 0.460 ± 0.008 vs. 0.320 ± 0.013 and 0.346 ± 0.013, all P< 0.01）. Conclusion High expression of β-catenin can raise proliferative activity of aging HSFs. Key words: Fibroblasts; Cell aging; β-catenin; Apoptosis; Genes, Bcl-2; Genes, Bax