全反式维A酸对恶性黑素瘤A375细胞上皮-间质转化相关分子表达的影响

Objective: To evaluate the effect of all-trans retinoic acid (ATRA) on the expression of epithelial-mesenchymal transition (EMT) -related molecules in human malignant melanoma A375 cells. Methods: Cultured A375 cells were divided into 4 groups: control-1 and -2 groups treated with Dulbecco's modified Eagle medium (DMEM) for 24 and 48 hours respectively, and ATRA-1 and ATRA-2 groups treated with DMEM containing 10 μmol/L ATRA for 24 and 48 hours respectively. After the treatment, real-time quantitative PCR was performed to determine the mRNA expression of EMT-related genes E-cadherin, N-cadherin, vimentin and β-catenin in the above 4 groups, Western blot analysis to determine the relative expression of the above proteins, and direct immunofluorescence study to assess the fluorescence intensity of E-cadherin and vimentin in the ATRA-1, ATRA-2 and control-1 groups. Statistical analysis was carried out by using two-way analysis of variance, one-way analysis of variance and least significant difference-t test. Results: Real-time quantitative PCR showed that the E-cadherin mRNA expression was significantly higher in the ATRA-1 group than in the control -1 group (F = 13.148, P < 0.05), and higher in the ATRA-2 group than in the control-2 group (F = 31.529, P < 0.05) ; the mRNA expression of N-cadherin, vimentin and β-catenin was significantly lower in the ATRA-1 group than in the control-1 group (P < 0.05), and lower in the ATRA-2 group than in the control-2 group (P < 0.05) ; the ATRA-2 group showed significantly increased mRNA expression of E-cadherin (F = 13.148, P < 0.05), but significantly decreased mRNA expression of the other 3 proteins compared with the ATRA-1 group (all P < 0.05) ; there was no significant difference in the mRNA expression of the above molecules between the control-1 and -2 groups (all P > 0.05). Western blot analysis showed that the protein expression of E-cadherin significantly increased, but the protein expression of N-cadherin, vimentin and β-catenin significantly decreased in the ATRA-1 and ATRA-2 groups compared with the control-1 group (all P < 0.05) ; compared with the ATRA-1 group, the ATRA-2 group showed significantly increased protein expression of E-cadherin (P < 0.05), but significantly decreased protein expression of N-cadherin, vimentin and β-catenin (all P < 0.05). Direct immunofluorescence study showed that the fluorescence intensity of E-cadherin was significantly higher in the ATRA-1 group and ATRA-2 group (6.23 ± 0.08, 10.37 ± 0.13, respectively) than in the control-1 group (2.37 ± 0.14, both P < 0.05), while the fluorescence intensity of vimentin was significantly lower in the ATRA-1 group and ATRA-2 group (15.17 ± 0.18, 10.29 ± 0.03, respectively) than in the control-1 group (50.16 ± 0.26, both P < 0.05), and the cells in the ATRA-1 group and ATRA-2 group transformed from spindle- to cobble-stone-like in shape. Conclusion: ATRA can up-regulate the expression of E-cadherin, down-regulate the expression of N-cadherin, vimentin and β-catenin in A375 cells, and may inhibit the EMT of A375 cells.