Abstract: Kang Jing*, Hrvoje Miletic, Guo Shulan. *Shandong University School of Medicine, Jinan 250012, China Corresponding author: Guo Shulan, Email: guoshulan@medmail.com.cn 【Abstract】 Objective To investigate the role of epidermal growth factor receptor （EGFR） in the growth and migration of melanoma. Methods A brain-metastatic human melanoma cell line H1, which had been developed in our laboratory, was used in this study. Some H1 melanoma cells were stably transfected with wild-type EGFR via lentiviral vectors to overexpress EGFR （H1EGFRwt）, and some H1 cells transfected with green fluorescent protein （GFP） served as the control group （H1GFP）. After transfection, the cells were sorted and purified. Then, scratch wound healing assay was performed to estimate the migratory activity of H1 cells; colony-forming assay was conducted to investigate the effects of EGFR on the survival ability and colony-forming ability of H1 cells, and resazurin reduction test was used to evaluate colony formation results; Western blot was carried out to measure the expressions of signaling pathways activated by EGFR. Statistical analysis was done by repeated measures analysis of variance （ANOVA） for the comparison of wound-area healing rate, and by paired t test for the comparison of metabolic activity, between the two groups of H1 cells by using SPSS software version 20.0. Results Flow cytometry showed that H1 cells were successfully transfected with EGFR and GFP respectively, and purified H1EGFRwt and H1GFP cells were obtained after cell sorting. The wound-area healing rate at 6, 18, 24 and 48 hours after epidermal growth factor （EGF） stimulation was 0.145 ± 0.066, 0.479 ± 0.096, 0.571 ± 0.198 and 0.672 ± 0.199 respectively for H1EGFRwt cells, 0.051 ± 0.036, 0.254 ± 0.038, 0.303 ± 0.077 and 0.498 ± 0.111 respectively for H1GFP cells. The degree of wound healing in H1EGFRwt cells was significantly higher than that in H1GFP cells （F = 68.49, df = 5, P < 0.05）. Colony-forming assay revealed that the average metabolic activity score of H1EGFRwt cells was significantly higher than that of H1GFP cells （92 225.2 ± 6 632.1 vs. 62 935.7 ± 8 159.2, t = 2.26, df = 9, P < 0.01）. Western blot showed that EGF induced the phosphorylation of downstream signaling proteins such as phosphatidylinositol-specific phospholipase C-γ （PLCγ）, signal transducer and activator of transcription 5 （STAT5） and 3 （STAT3） in H1EGFRwt cells, but not in H1GFP cells. In addition, the expressions of phosphorylated serine/threonine protein kinase （pAKT） and mitogen-activated protein kinase （pMAPK） were significantly higher in H1EGFRwt cells than in H1GFP cells after EGF stimulation. Conclusions EGFR may play an important role in the metastasis of H1 melanoma cells, and might serve as a target in the treatment of metastatic melanoma.

Key words: Melanoma, Receptor, epidermal growth factor, Epidermal growth factor