正常人黑素细胞MITF对酪氨酸酶相关蛋白的转录调控研究

Effect of MITF Transcriptional Control on Family of Tyrosinase-related Proteins in Normal Human Melanocytes

目的研究正常人黑素细胞小眼畸形相关转录因子(MITF)对与黑素生成密切相关的酪氨酸酶相关蛋白(TRPs)家族的转录调控。方法应用小RNA干扰技术(慢病毒感染),对培养的原代正常人黑素细胞进行MITF基因沉默,然后应用实时定量PCR和免疫印迹方法,分别检测MITF基因沉默前后,MITF、酪氨酸酶(TYR)、酪氨酸酶相关蛋白1(TYRP1)和酪氨酸酶相关蛋白2(TYRP2)的基因与蛋白表达水平。结果正常人黑素细胞具有显著表达的MITF、TYR、TYRP1和TYRP2;MITF基因沉默后,MITF、TYR和TYRP1的基因表达水平分别下降2倍(P=0.001)、1.5倍(P=0.05)和5倍(P=0.005),TYRP2的表达水平上调3倍(P=0.004);而MITF基因沉默后的MITF、TYR、TYRP1的蛋白表达也显著下降,TYRP2蛋白显著升高。结论 黑素细胞TRPs家族与MITF转录调控密切相关,TYRP2可能存在不依赖于MITF的转录调控。

Objective To analyze the transcriptional control of microphthalmia transcription factor （MITF）on the tyrosinase related proteins which is closely related to melanogenesis in normal human melanocytes. Methods Normal human melanocytes from healthy chil- dren＇s circumcision were cultured in melanoeyte Hul6 selective medium. MITF was knocked down by using MITF siRNA interference. Real time quantitative PCR and Western blot were used to investigate the gene and proteins expression of MITF, tyrosinase（TYR） , tyrosi- nase related protein 1 （TYRP1）and tyrosinase related protein 2 （TYRP2）. Results The MITF, TYR, TYRP1 and TYRP2 were signifi- cant expressed in normal human melanocytes. After MITF was knocked down, we found that the expression of TYR and TYRP1 were dra- matically decreased and the expression of TYRP2 was increased both in mRNA and protein expression. The mRNA expression of MITF, TYR and TYRPI were decreased about 2 folds （P = 0. 001 ） , 1.5 folds （ P = 0.05 ） and 5 folds （ P = 0. 005 ） respectively, while TYRP2 was increased dramatically 3 folds （P = 0. 004）. Conclusion The tyrosinase related protein （TRPs） was closely related to transcription- al control of MITF. TYRP2 may be regulated by MITF through different pathway compared to TYR and TYRP1.