关键词: 黑色素瘤, E2F6转录因子, β连环素, 细胞增殖, 细胞凋亡, A375细胞

Abstract: 【Abstract】 Objective To determine the expression of the E2F6 transcription factor in human malignant melanoma tissues and cell lines, and to evaluate the effect of E2F6 on proliferation, migration and invasion of a malignant melanoma cell line A375. Methods Frozen tissues and paraffin-embedded tissue sections were collected from 50 cases of cutaneous malignant melanoma and 30 cases of pigmented nevus in Department of Dermatology, Chongqing Traditional Chinese Medicine Hospital from January 2012 to December 2017. Quantitative reverse transcription-PCR （qRT-PCR） was performed to determine the mRNA expression of E2F6 in the malignant melanoma and pigmented nevus tissues, as well as in 7 malignant melanoma cell lines （HM, A375, WM451, WM35, SK-MEL-1, Hs-695T and MDA-MB-435s） and pigmented nevus cells, and immunohistochemical study and Western blot analysis were conducted to determine the protein expression of E2F6 and β-catenin in the malignant melanoma tissues. An E2F6-inhibiting plasmid and a control plasmid were separately transfected into A375 cells by using a liposome-mediated transfection method, and the E2F6 gene-knockdown efficiency was verified by qRT-PCR and Western blot analysis. Cell counting kit-8 （CCK8） assay, soft-agar plate cloning assay, Transwell migration and invasion assays and 3D cell culture assay were conducted to evaluate the effect of E2F6 gene knockdown on the proliferation, migration and invasion of A375 cells, flow cytometry was performed to detect the cell cycle and apoptosis rate, and Western blot analysis was conducted to determine the protein expression of total β-catenin, activated β-catenin, c-Myc and cyclin D1. The comparison between two groups was carried out by t test, the comparison among several groups by one-way analysis of variance, and multiple comparisons by least significant difference t test; Pearson correlation coefficient was used to analyze the correlation between E2F6 and β-catenin expression in cutaneous malignant melanoma. Results The E2F6 mRNA expression was significantly higher in the 7 malignant melanoma cell lines than in the pigmented nevus cells （all P < 0.001）. qRT-PCR showed that the relative mRNA expression of E2F6 was significantly higher in the cutaneous malignant melanoma tissues （0.000 55 ± 0.000 17） than in the pigmented nevus tissues （0.000 18 ± 0.000 09, t = 3.22, P < 0.001）. Both the immunohistochemical study and Western blot analysis showed significantly increased E2F6 protein expression, but decreased β-catenin protein expression in the cutaneous malignant melanoma tissues compared with the pigmented nevus tissues （all P < 0.001）. Correlation analysis showed that E2F6 protein expression was negatively correlated with β-catenin expression in the malignant melanoma tissues （immunohistochemical study: r = -0.56, Western blot analysis: r = -0.63, both P < 0.01）. After knockdown of the E2F6 gene in A375 cells, the mRNA and protein expression of E2F6 was significantly lower in the E2F6 inhibition group than in the control group （t = 3.38, 2.76 respectively, both P < 0.001）. CCK8 assay showed that the cellular proliferative ability was significantly lower in the E2F6 inhibition group than in the control group （t = 4.58, P < 0.01） 48 hours after transfection; soft-agar plate cloning assay showed that the colony-formation ratio was significantly lower in the E2F6 inhibition group than in the control group （t = 2.26, P < 0.001）; Transwell migration and invasion assays showed that the number of cells crossing the chamber was significantly lower in the E2F6 inhibition group （165 ± 23, 96 ± 11 respectively） than in the control group （376 ± 22, 315 ± 31, t = 3.14, 2.12, respectively, both P < 0.01）; 3D cell culture assay showed that the cell morphology markedly changed, and the invasive pseudopodia disappeared in the E2F6 inhibition group. Flow cytometry revealed that the proportion of cells at G0 - G1 phase and apoptosis rate were significantly higher in the E2F6 inhibition group than in the control group （both P < 0.001）. Western blot analysis showed significantly decreased protein expression of β-catenin, activated β-catenin and its downstream target proteins c-Myc and cyclin D1, but significantly increased protein expression of P21 in the E2F6 inhibition group compared with the control group （all P < 0.001）; additionally, the E2F6 inhibition group showed significantly decreased protein expression of epithelial-mesenchymal transition-related molecules vimentin and N-cadherin, but significantly increased expression of E-cadherin compared with the control group （all P < 0.001）. Conclusions The E2F6 transcription factor is highly expressed in malignant melanoma. Knockdown of the E2F6 gene in A375 cells can inhibit cell proliferation, migration and invasion by antagonizing the β-catenin signaling pathway.

Key words: Melanoma, E2F6 transcription factor, beta Catenin, Cell proliferation, Apoptosis, A375 cell