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

Effect of all-trans retinoic acid on the expression of epithelial-mesenchymal transition-related molecules in human malignant melanoma A375 cells

目的研究全反式维A酸(ATRA)对人恶性黑素瘤A375细胞中上皮-间质转化(EMT)相关分子表达的影响。方法用含10μmol/L ATRA的DMEM培养基和DMEM培养基分别处理A375细胞24和48 h作为ATRA-1组和ATRA-2组、对照1组和对照2组,采用实时定量PCR法检测EMT相关基因上皮钙黏着蛋白、神经钙黏着蛋白、波形蛋白、β联蛋白mRNA的表达。Western印迹法检测ATRA-1组、ATRA-2组、对照1组中上述蛋白的相对表达量,直接免疫荧光法检测上皮钙黏着蛋白和波形蛋白的荧光强度。统计分析采用两因素方差分析、单因素方差分析及LSD-t检验。结果ATRA-1组和ATRA-2组分别与对照1组和对照2组相比,上皮钙黏着蛋白mRNA的表达均显著增加(F=13.148、31.529,P<0.05),而神经钙黏着蛋白、波形蛋白、β联蛋白mRNA的表达均显著降低(均P<0.05);ATRA-2组上皮钙黏着蛋白mRNA的表达显著高于ATRA-1组(F=13.148,P<0.05),而其他3种蛋白mRNA的表达显著低于ATRA-1组(均P<0.05);对照1组与对照2组上述蛋白的mRNA表达差异无统计学意义(均P>0.05)。Western印迹法显示,与对照1组相比,ATRA-1组和ATRA-2组上皮钙黏着蛋白表达上调,而神经钙黏着蛋白、波形蛋白、β联蛋白表达均下调(均P<0.05);与ATRA-1组相比,ATRA-2组上皮钙黏着蛋白表达上调(P<0.05),神经钙黏着蛋白、波形蛋白、β联蛋白表达下调(均P<0.05)。直接免疫荧光显示,ATRA-1组、ATRA-2组上皮钙黏着蛋白的荧光强度(6.23±0.08、10.37±0.13)显著高于对照1组(2.37±0.14,均P<0.05),而波形蛋白的荧光强度(15.17±0.18、10.29±0.03)显著低于对照1组(50.16±0.26,均P<0.05),抑制上皮向间质转化。结论ATRA可上调A375细胞中上皮钙黏着蛋白的表达,降低神经钙黏着蛋白、波形蛋白、β联蛋白的表达,可能抑制A375细胞发生EMT现象。

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 10μmol/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 andβ-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 andβ-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 andβ-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 andβ-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 andβ-catenin in A375 cells,and may inhibit the EMT of A375 cells.