Mechanism underlying the inhibitory effect of overexpression of tumor suppressor gene DKK3 on migration and invasion of human cutaneous malignant melanoma cells

Abstract

Objective To investigate the expression of DKK3 in human cutaneous malignant melanoma cells and tissues, and to evaluate the effect of transfection with DKK3 gene on migration and invasion of a malignant melanoma cell line A375. Methods Western blot analysis was performed to measure the relative expression of DKK3 in human cutaneous melanoma cell lines HM, A375, WM451, SK-MEL-1, Hs-695T, MDA-MB-435s and WM35, as well as pigmented nevus tissues. Real-time fluorescence-based quantitative PCR (RT-PCR) was conducted to determine the mRNA expression of DKK3 in 58 melanoma tissues (including primary melanoma and metastatic melanoma) and 30 pigmented nevus tissues from Chongqing Traditional Chinese Medicine Hospital between August 2014 and June 2017. The pcDNA3.1 (+) -Flag-Vector (control group) and pcDNA3.1 (+) -Flag-DKK3 (transfection group) were transfected into A375 melanoma cells separately. RT-PCR and Western blot analysis were performed to verify the overexpression of DKK3, and to evaluate the effect of DKK3 overexpression on the expression of molecules related to the migration and invasion of melanoma cells. Cell scratch assay, Transwell migration and invasion assay were conducted to assess the effect of DKK3 on the migration and invasion of A375 cells. Statistical analysis was done by a two-sample t-test for comparisons between two groups, one-way analysis of variance (ANOVA) for intergroup comparison, and least significant difference (LSD) -t test for multiple comparisons with the SPSS 13.0 software. Results DKK3 protein was absent or lowly expressed in the human melanoma cell lines, but highly expressed in the pigmented nevus tissues. There were significant differences in the mRNA expression of DKK3 among the primary melanoma tissues (2-ΔΔCt:[0.325 ± 0.150] × 10-3) , metastatic melanoma tissues ([0.142 ± 0.210] × 10-3) and pigmented nevus tissues ([0.634 ± 0.120] × 10-3, F = 46.57, P < 0.05) . In addition, the mRNA expression of DKK3 was significantly lower in the metastatic melanoma tissues than in the primary melanoma tissues and pigmented nevus tissues (LSD-t = 2.48, 3.12, both P < 0.05) . After transfection with DKK3, cell scratch assay showed that the migration rate was significantly lower in the transfection group (22.11% ± 5.11%) than in the control group (54.36% ± 23.22%, t = 2.36, P < 0.001) . Transwell migration and invasion assay revealed that the number of A375 cells crossing the Transwell chamber was significantly lower in the transfection group (265 ± 33, 76 ± 18 respectively) than in the control group (429 ± 41, 135 ± 21 respectively; t = 1.24, 1.35 respectively, both P < 0.001) . After overexpression of DKK3 in the A375 cells in the transfection group, the mRNA and protein expression of E-cadherin were up-regulated, while the mRNA and protein expression of N-cadherin, vimentin, matrix metalloproteinase 2 (MMP2) , MMP7 and MMP11 were down-regulated compared with the control group. Conclusions The expression of DKK3 is down-regulated in the melanoma cell lines and tissues, and the migration and invasion of A375 cells are markedly inhibited by overexpression of DKK3. DKK3 may be a target for inhibiting the metastasis of cutaneous malignant melanoma. Key words: Melanoma; Genes, tumor suppressor; Cell movement; Invasion; Dickkopf-related protein 3; A375 cells