Figure 9. Knockdown of β-catenin by SiRNA prevented a decrease of both Na_V1.5 expression and SCN5a promoter activity induced by H₂O₂.

HL-1 cells were transfected with 100 nM β-catenin and scramble SiRNA, respectively, for 48 hours without or with 48 hours 50 μM H₂O₂ treatment. Total Protein and RNA were extracted from these cells. Western blot and real time-PCR were performed. (A) 100 nM β-catenin SiRNA significantly knocked down β-catenin expression at both protein and mRNA levels (p<0.01) and significantly increased Na_V1.5 protein and mRNA (p<0.01), compared to the 100 nM scramble SiRNA group. (B) Knockdown of β-catenin completely abolished suppressive effects of 50 μM H₂O₂ for 48 hours on Na_V1.5 expression at both protein and mRNA levels. (C) Luciferase promoter assays were performed on the protein extracts from transfected Hela cells treated with β-catenin or scramble SiRNA or treated with or without 50 μM H₂O₂ for 48 hours. The results showed that β-catenin SiRNA significantly increased SCN5a promoter activity (p<0.05), compared to the scramble SiRNA control group and that H₂O₂ significantly inhibited SCN5a promoter activity (p<0.01), compared to the control group. In order to determine if H₂O₂ effects on the SCN5a promoter activity is related to β-catenin, luciferase promoter assays were performed on the protein extracts from Hela cells transfected with β-catenin or scramble SiRNA and these cells were cultured in the medium containing 50 μM H₂O₂. The results showed that b-catenin SiRNA completely abolished the H₂O₂ suppressive effects, compared to the scramble group. *p<0.05 and **p<0.01; n=3 batches of HL-1 or HeLa cells from 3 independent experiments carried on separate occasions for each group.