非转移性黑素瘤糖蛋白基因沉默抑制黑素小体形成的机制探讨

Study on the mechanism of suppression of GPNMB gene silencing on melanosome formation

目的:探讨非转移性黑素瘤糖蛋白[glycoprotein(transmembrane)nonmetastatic melanoma proteinb,GPNM]B基因沉默抑制黑素小体形成的作用机制。方法:以黑素细胞系PIG1为研究对象,采用小干扰RNA(siRNA)干扰法下调GPNMB基因的表达,以实时定量反转录(RT)-PCR和Western blot法检测GPNMB-siRNA转染对GPNMB、酪氨酸酶(Tyr)、酪氨酸相关蛋白1(Trp1)、OA1和Pmel17的影响,进一步采用Tyr-siRNA转染PIG1细胞下调Tyr基因的表达以验证GPNMB与Tyr之间的相互作用,并检测GPNMB基因沉默对小眼畸形相关转录因子(MITF)的影响。结果:GPNMB-siRNA转染对GPNMB、Tyr、Trp1、Pmel17和OA1等黑素小体蛋白的表达均有显著抑制作用;GPNMB与Tyr之间无相互作用;GPNMB-siRNA转染或中波紫外线(UVB)单独作用能够上调MITF的表达,但两者共同作用却能显著抑制MITF的表达。结论:GPNMB基因沉默对黑素小体形成的抑制作用不依赖于MITF。

Objective： To explore the mechanism of suppression of glycoprotein （transmembrane） nonmetastatic melanoma pro- tein B （GPNMB） gene silencing on melanosome formation. Methods： GPNMB siRNA was used to down-regulate GPNMB ex- pression in PIG1 melanocytes. Real-time quantitative RT-PCR and Western blot were employed to determine GPNMB-siRNA transfeetion on the expression levels of GPNMB, tyrosinase （Tyr）, tyrosinase related protein 1 （Trp1）, OA1 and Pmel17. To con- firm the interaction between GPNMB and Tyr, down regulation of Tyr mRNA was achieved by transfection of Tyr with Tyr siRNA in PIG1 melanocytes. The effect of GPNMB gene silencing on microphthalmia-associated transcription factor （MITF） ex- pression was also determined. Results： The expression of GPNMB, Tyr, Trp1, OA1 and Pmel17 were markedly suppressed by GPNMB transfection. No interaction between GPNMB and Tyr was observed. Ultraviolet B radiation or GPNMB transfection alone up-regulated MITF expression in PIG1 melanocytes. But the combination of ultraviolet B radiation and GPNMB transfec- tion markedly inhibited MITF expression in PIG1 melanocytes. Conclusion： The inhibitory effect of GPNMB gene silencing on melanosome formation is MITF-independent.