Wnt5A gene silencing in primary human melanocytes
10.3760/cma.j.issn.0412-4030.2017.10.003
- VernacularTitle:原代人黑素细胞Wnt5A基因沉默的研究
- Author:
Qianya SU
1
,
2
;
东南大学附属中大医院皮肤科,210009南京
;
Tong LIN
;
Mengli ZHANG
;
Lin PENG
Author Information
1. 210042南京,中国医学科学院北京协和医学院皮肤病医院激光科
2. 东南大学附属中大医院皮肤科,210009南京
- Keywords:
Melanocytes;
RNA,small interfering;
Gene silencing;
Wnt5A
- From:
Chinese Journal of Dermatology
2017;50(10):706-709
- CountryChina
- Language:Chinese
-
Abstract:
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.Realtime 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 expression 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 expression (all P ＜ 0.05),as well as the best transfection efficiency.The mRNA expression 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 expression (all P ＜ 0.05),as well as the best transfection efficiency.The mRNA expression 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 expression 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 expression 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 expression 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.
