关键词: 黑素细胞, 细胞增殖, 细胞迁移分析, 细胞黏附, Fam114A1, A375细胞, 黑素合成

Abstract: 【Abstract】 Objective To preliminarily evaluate the effect of Fam114A1 on the biological function of melanocytes. Methods A375 human melanoma cells was used to construct stably Fam114A1-overexpressing or -inhibited cell line by lentiviral transfection, namely overexpression group and expression inhibition group respectively, and A375 cells transfected with an empty lentivirus served as control group. Real-time fluorescence-based quantitative PCR was performed to evaluate effect of Fam114A1 on the mRNA expression of melanin synthesis-related genes tyrosinase （TYR）, tyrosinase-related protein 1 （TYRP1）, premelanosome protein （PMEL）, microphthalmia-associated transcription factor （MITF） and dopachrome isomerase （DCT）, Western blot analysis was conducted to determine the protein expression of TYR and MITF, methyl thiazol tetrazolium （MTT） assay, Transwell migration and adhesion assays were conducted to assess the effect of Fam114A1 on cellular proliferative activity, migratory and adhesive ability of A375 cells respectively. Statistical analysis was carried out by using one-way analysis of variance and Dunnett-t test. Results Fluorescence microscopy showed that lentivirus-based transfection efficiency was about 90% in the 3 groups. Compared with the control group （0.850 ± 0.120）, the protein expression of Fam114A1 significantly increased in the overexpression group （1.507 ± 0.170, t = 5.888, P = 0.001）, but significantly decreased in the expression inhibition group （0.397 ± 0.120, t = 4.065, P = 0.007）, suggesting that the stably Fam114A1-overexpressing or -inhibited A375 cell line was successfully constructed. Real-time fluorescence-based quantitative PCR and Western blot analysis showed that the mRNA and protein expression of TYR and MITF were significantly lower in the expression inhibition group than in the control group （all P < 0.01）, but did not differ between the overexpression group and control group （all P > 0.05）. Compared with the control group, the expression inhibition group showed significantly increased cellular proliferative activity and adhesive ability （P = 0.009, 0.001, respectively）, but significantly decreased migratory ability （P = 0.005）, while the overexpression group only showed significantly increased migratory ability （P = 0.021）. Conclusions Fam114A1 can affect the proliferative activity, migratory and adhesive abilities of A375 cells, and the expression of melanin synthesis-related proteins TYR and MITF in A375 cells. Fam114A1 may be a functional protein involved in regulating the biological activity of melanocytes.

Key words: Melanocytes, Cell proliferation, Cell migration assays, Cell adhesion, Fam114A1, A375 cells, Melanin synthesis