Abstract: 【Objective】 The objective of this study is to explore whether GPNMB affects melanin synthesis in the melanocytes by regulating the levels of MITF and its downstream pigmentation-related genes, further clarify the specific mechanism of GPNMB in the melanin genesis in the melanocytes to provide a theoretical foundation.【Method】Firstly, the mouse GPNMB nucleic acid sequences was searched from NCBI. After analysis of the sequences of GPNMB and the lentiviral expression vector, the Sal I and Xba I restriction sites was selected. Then, the GPNMB full-length primers containing SalI and XbaI restriction sites was designed and the GPNMB full-length sequence containing the restriction site was cloned, and finally, the T vector was ligated and sequenced. The constructed T vector plasmid was extracted and doubly digested, followed by ligating with the lentiviral expression vector containing green fluorescence protein (GFP) and then sequenced. A large number of endotoxin-free GPNMB lentivirus eukaryotic expression vectors were collected according to the instruction of the plasmid midiprep system. Furthermore, mouse melanocytes was cultured in vitro and transfected using Lipofectamine 2000 reagent according to the manufacturer’s guidelines when it was in the logarithmic growth phase. Forty-eight hours after transfection, the morphological changes and green fluorescent protein expression levels of melanocytes was observed. Then, the cells were harvested and the transfection efficiency and melanin content of melanocytes were tested. The relative expression levels of PMEL, MITF, TYR, TYRP1, TYRP2 and OA1 were qualitatively and quantitatively analyzed by real-time quantitative PCR (qRT-PCR) and Western blot. 【Result】 The morphological characteristics of melanocytes in the experimental group and empty vector group had no obvious change compared with the normal group. The results of green fluorescent protein (GFP) in both the experimental group and the empty vector group showed that the transfection efficiency in 5 μg DNA / well was the highest. GPNMB mRNA and protein levels in the experimental group were extremely significantly higher than the empty vector group and normal group. Compared with the empty vector group, the melanin content in the experimental group increased by 1.34 times (P＜0.01). The qRT-PCR results showed that the relative expression levels of PMEL, TYRP1 and TYRP2 mRNA in experimental group was 1.59 (P＜0.05), 2.35 (P＜0.01) and 1.60 times (P＜0.01) of that in empty vector group, respectively; However, the relative expression level of MITF mRNA was significantly reduced by 2.25 times (P＜0.01); TYR and OA1 mRNA increased 1.65 and 1.50 times, respectively, but not significantly. Furthermore, the western blot results showed that TYRP2 protein was significantly increased by 4.35 times (P＜0.01); TYR protein was significantly increased by 1.23 times (P＜0.01); OA1 protein was significantly increased by 1.31 times (P＜0.05); By contrast, MITF protein was significantly reduced by 1.59 times (P＜0.01); TYRP1 protein had no significantly change.【Conclusion】Over-expression of GPNMB increased the expression of PMEL, TYR, TYRP1, TYRP2 and OA1, but reduced the expression of MITF. It was speculation that GPNMB may affect the melanin synthesis by regulating the expression of PMEL, TYR, TYRP1, TYRP2 and OA1 in a MITF-independent fashion.

Key words: GPNMB, MITF, melanocyte, melanin synthesis