中华皮肤科杂志 ›› 2021, Vol. 54 ›› Issue (1): 50-55.doi: 10.35541/cjd.20200292

• 黑素瘤·论著 • 上一篇    下一篇

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

向桂琼1    樊卓1    党云1    郭坤2    耿松梅2   

  1. 1西安医学院第一附属医院皮肤科  710077；2西安交通大学医学院第二附属医院皮肤病院  710004
  • 收稿日期:2020-03-25 修回日期:2020-08-30 发布日期:2021-01-05
  • 通讯作者: 耿松梅；郭坤 E-mail:gsm312@yahoo.com; 05322987612guoguo@163.com
  • 基金资助:
    国家自然科学基金（81372912、51707149）；西安交通大学基本科研业务费（xjj2017071）

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

Xiang Guiqiong1, Fan Zhuo1, Dang Yun1, Guo Kun2, Geng Songmei2   

  1. 1Department of Dermatology, The First Affiliated Hospital of Xi′an Medical University, Xi′an 710077, China; 2Department of Dermatology, The Second Affiliated Hospital of Xi′an Jiaotong University, Xi′an 710004, China
  • Received:2020-03-25 Revised:2020-08-30 Published:2021-01-05
  • Contact: Geng Songmei; Guo Kun E-mail:gsm312@yahoo.com; 05322987612guoguo@163.com
  • Supported by:
    National Natural Science Foundation of China（81372912, 51707149）; Fundamental Research Fund of Xi′an Jiaotong University（xjj2017071）

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摘要： 【摘要】 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10 μmol/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。

关键词: 黑色素瘤, 上皮-间质转化, 钙黏着糖蛋白类, 波形蛋白, β连环素, 全反式维A酸

Abstract: 【Abstract】 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.

Key words: Melanoma, Epithelial-mesenchymal transition, Cadherins, Vimentin, beta Catenin, All-trans retinoic acid

引用本文

向桂琼 樊卓 党云 郭坤 耿松梅. 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[J]. 中华皮肤科杂志, 2021,54(1):50-55. doi:10.35541/cjd.20200292

Xiang Guiqiong, Fan Zhuo, Dang Yun, Guo Kun, Geng Songmei. Effect of all-trans retinoic acid on the expression of epithelial-mesenchymal transition-related molecules in human malignant melanoma A375 cells[J]. Chinese Journal of Dermatology, 2021, 54(1): 50-55.doi:10.35541/cjd.20200292