原代人黑素细胞Wnt5A基因沉默的研究

Wnt5A gene silencing in primary human melanocytes

目的 优化Wnt5A特异性siRNA转染原代人黑素细胞的实验条件，建立沉默Wnt5A基因的模型。方法 培养原代人黑素细胞，将不同浓度（0、20.0、33.3、40.0、60.0 nmol/L）的阳性参照基因GAPDH特异性siRNA通过脂质体法介导转染原代人黑素细胞，实时荧光定量PCR（qPCR）筛选转染效率最高的siRNA浓度；合成3条Wnt5A-siRNA（Wnt5A-siRNA-793、Wnt5A-siRNA-943、Wnt5A-siRNA-1743），分别转染原代人表皮黑素细胞，qPCR法选择干扰特异性最佳的Wnt5A-siRNA；用最佳Wnt5A-siRNA转染原代人黑素细胞，分别于培养24、48、72 h提取总mRNA及总蛋白，通过qPCR及Western印迹法确定转染效率最高的时间点。结果 0、20.0、33.3、40.0、60.0 nmol/L GAPDH-siRNA转染原代人黑素细胞后，GAPDH mRNA相对表达量分别为1.009 ± 0.161、0.086 ± 0.010、0.140 ± 0.016、0.285 ± 0.095、0.012 ± 0.007，组间比较，差异有统计学意义（F = 69.469，P 〈 0.05）。60 nmol/L siRNA组GAPDH表达低于其他各组（均P 〈 0.05），干扰效率最佳；各Wnt5A-siRNA组及空白组Wnt5A mRNA相对表达量分别为0.331 ± 0.010、2.229 ± 0.029、0.078 ± 0.006、1.000 ± 0.024，差异有统计学意义（F = 7 006.094，P 〈 0.05）。Wnt5A-siRNA-1743组目标分子表达量低于其他3组（均P 〈 0.05），干扰效率最佳。Wnt5A-siRNA-1743转染原代人表皮黑素细胞后24、48、72 h及空白组 Wnt5A mRNA相对表达量分别为0.396 ± 0.002、0.026 ± 0.008、0.131 ± 0.079、1.025 ± 0.276，组间比较，差异有统计学意义（F = 29.215，P 〈0.05），干扰后48 h低于干扰后24、72 h及空白组（均P 〈 0.05）；Wnt5A蛋白相对表达量分别为112.798 ± 0.218、77.765 ± 0.415、30.540 ± 0.219、130.025 ± 0.158，组间比较，差异有统计学意义（F = 79 122.889，P 〈 0.05），干扰后72 h低于干扰后24、48 h及空白组（均P 〈 0.05）。结论 成功建立siRNA沉默人黑素细胞Wnt5A基因的模型。

Objective To optimize experimental conditions for transfecting primary human melanocytes with Wnt5A-specific siRNA, and to establish the Wnt5A gene silencing model. Methods Primary human melanocytes（PHM）were cultured in vitro. Positive control GAPDH-siRNAs at different concentrations of 0, 20.0, 33.3, 40.0 and 60.0 nmol/L were transfected into PHM by using liposomes. Real-time fluorescence-based quantitative PCR （qPCR） was performed to select the optimal concentration of siRNA with the highest transfection efficiency. Three Wnt5A-siRNAs including Wnt5A-siRNA-793, Wnt5A-siRNA-943 and Wnt5A-siRNA-1743 were constructed and transfected into PHM separately, and qPCR was conducted to select the most specific Wnt5A-siRNA. Then, the most specific Wnt5A-siRNA was transfected into PHM, and the total mRNA and total protein were extracted after 24-, 48- and 72-hour treatment. qPCR and Western blot analysis were performed to determine the optimal time with the highest transfection efficiency. Results There were significant differences in the mRNA of GAPDH among cells transfected with GAPDH-siRNAs at different concentrations of 0, 20.0, 33.3, 40.0 and 60.0 nmol/L （1.009 ± 0.161, 0.086 ± 0.010, 0.140 ± 0.016, 0.285 ± 0.095, 0.012 ± 0.007 respectively; F = 69.469, P 〈 0.05）. Additionally, the 60-nmol/L GAPDH-siRNA group showed the lowest GAPDH mRNA （all P 〈 0.05）, as well as the best transfection efficiency. The mRNA of Wnt5A differed in the Wnt5A-siRNA-793 group, Wnt5A-siRNA-943 group, Wnt5A-siRNA-1743 group and the blank control group （0.331 ± 0.010, 2.229 ± 0.029, 0.078 ± 0.006 and 1.000 ± 0.024 respectively; F = 7 006.094, P 〈 0.05）, and the Wnt5A-siRNA-1743 group showed the lowest Wnt5A mRNA （all P 〈 0.05）, as well as the best transfection efficiency. The mRNA of Wnt5A differed among the Wnt5A-siRNA-1743 group after 24-, 48- and 72-hour treatment and the blank control group （0.396 ± 0.002, 0.026 ± 0.008, 0.131 ± 0.079, 1.025 ± 0.276 respectively; F = 29.215, P 〈 0.05）, so did the protein of Wnt5A （112.798 ± 0.218, 77.765 ± 0.415, 30.540 ± 0.219, 130.025 ± 0.158 respectively; F = 79 122.889, P 〈 0.05）. Moreover, the Wnt5A mRNA was significantly lower in the Wnt5A-siRNA-1743 group at 48 hours compared with that at 24 and 72 hours and the blank control group（all P 〈 0.05）, while the Wnt5A protein was significantly lower in the Wnt5A-siRNA-1743 group at 72 hours compared with that at 24 and 48 hours and the blank control group（all P 〈 0.05）. Conclusion The siRNA-mediated Wnt5A gene silencing model of human melanocytes is established successfully.