Effect of ADAR1 gene silencing in HaCaT cells on Wnt11 expression and tyrosinase activity in human A375 melanoma cells

Abstract

Objective To estimate the influence of ADAR1 gene,which is considered to be responsible for the pathogenesis of dyschromatosis symmetrica hereditaria,on Wnt1 1 expression and tyrosinase activity.Methods Some cultured HaCaT cells were equally divided into four groups:control group remaining untreated,three experimental groups transfected with three different ADAR1-specific shRNAs respectively.Then,Western blot was performed to quantify the expression of Wnt11 protein in HaCaT cells so as to select the most potent shRNA.Some human A375 melanoma cells were cocultured with untransfected HaCaT cells (normally expressing ADAR1 and Wnt1 1 proteins) or HaCaT cells transfected with the selected specific shRNA (lowly expressing ADAR1 and Wnt11 proteins).Thereafter,cell appearance was observed using inverted microscopy at 24,48 and 72 hours,and tyrosinase activity was estimated at 48 hours.Results As Western blot showed,the expression of Wnt 11 protein was significantly lower in the three ADAR1-silenced experimental groups than in the control group.The number of dendritic protrusions at the junction sites between HaCaT cells and A375 cells was significantly decreased,together with a significant reduction in tyrosinase activity (absorbance value:0.0168 ± 0.0069 vs.0.0490 ± 0.0132,P ＜0.01),in A375 cells cocultured with transfected HaCaT cells compared with those cocultured with normal control HaCaT cells.Conclusion ADAR1 gene silencing in HaCaT cells can attenuate the expression of Wnt11 protein,and affect tyrosinase activity in A375 cells. Key words: Genes, ADAR1; Wnt11; Melanocytes; Keratinocytes