HaCaT细胞ADAR1基因沉默对Wnt11表达和A375细胞酪氨酸酶活性的影响

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

目的探讨遗传性对称性色素异常症致病基因ADARl对Wntll表达和酪氨酸酶活性的影响。方法将HaCaT细胞等细胞数量分为对照组、实验1组、实验2组、实验3组。用3种针对ADARl基因的shRNA质粒分别沉默3个实验组中HaCaT细胞的ADARI基因，用Western印迹法检测4个组HaCaT细胞相关蛋白的表达水平。建立HacaT细胞与黑素瘤细胞A375共培养模型，对实验组（HacaT细胞中的ADARI和Wntll蛋白低表达）与对照组（HaCaT细胞中的ADARl和Wntll蛋白正常表达）的细胞形态和酪氨酸酶活性进行比较。结果蛋白印迹法发现，ADARl基因沉默的HaCaT细胞中的Wntll蛋白条带灰度值明显低于正常HaCaT细胞中的Wntll蛋白条带灰度值，证实HaCaT细胞中的ADARl基因沉默后，Wntll蛋白的表达水平明显下降。在共培养模型中，实验组HaCaT细胞与A375细胞连接处的树突状突起明显少于对照组，实验组A375细胞的酪氨酸酶活性与对照组相比显著降低，减去空白组A值后，实验组A值为0．0168±0．0069，对照组为0．0490±0．0132，P〈0．01。结论HaCaT细胞中ADARl基因沉默可降低Wntll的表达，并影响A375细胞酪氨酸酶活性。

Objective To estimate the influence of ADAR1 gene, which is considered to be responsible for the pathogenesis of dysehromatosis symmetrica hereditaria, on Wntl 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 ADARl-specific shRNAs respectively. Then, Western blot was performed to quantify the expression of Wntl 1 protein in HaCaT cells so as to select the most potent shRNA. Some human A375 melanoma cells were cocuhured with untransfected HaCaT cells （normally expressing ADAR1 and Wntll proteins） or HaCaT cells transfected with the selected specific shRNA （lowly expressing ADAR1 and Wntll 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 Wntl 1 protein was significantly lower in the three ADARl-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 cocuhured with transfected HaCaT cells compared with those cocuhured with normal control HaCaT cells. Conclusion ADAR1 gene silencing in HaCaT cells can attenuate the expression of Wntl 1 protein, and affect tyrosinase activity in A375 ceils.