Zhao Xiaozhen, Wu Zhonghua, Xu Zhenye, Wang Zhongqi, Wu Ji, Su Wan
Department of Oncology Section of Longhua Hospital, Cancer Institute of Chinese Medicine, and Science and Technology Experiment Center, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China.
Zhongguo Fei Ai Za Zhi. 2011 Jun;14(6):467-71. doi: 10.3779/j.issn.1009-3419.2011.06.01.
Our previous in vivo experiment (paper has been published) has confirmed Feiyanning's regulation effect on epithelial cell markers Alpha-catenin, beta-catenin, E-Cadherin and mesenchymal cell markers N-cadherin, Fibronectin, vimentin. Based on previous study, the objective of this study is to further investigate the expression of epithelial-mesenchymal cell marker gene and protein intervened by Feiyanning in highly metastatic lung cancer cells 95-D in vitro.
Human highly metastatic lung cancer 95-D cells were treated with different concentrations of Feiyanning in vitro, and then Real-time PCR and Western blot methods were used to detect mRNA and protein expression of epithelial-mesenchymal cell marker factor Alpha-catenin, beta-catenin, E-Cadherin, N-cadherin, Fibronectin and vimentin.
Real-time PCR results showed that compared with the control group, Feiyanning at 20% concentration remarkably up-regulated the expression of Alpha-catenin (P<0.05), Feiyanning at 20% and 25% concentrations remarkably up-regulated the expression of E-Cadherin (P<0.05, P<0.01), Feiyanning had no significantly effect on beta-catenin at any concentration, 5% and 10% Feiyanning at 5% and 10% concentrations significantly dow-regulated the expression of mesenchymal cell marker vimentin factor (P<0.01), Feiyanning had no regulatory role on N-cadherin and Fibronectin at any concentration. Western blot results showed that Feiyanning at 5%, 10%, 15%, 20% and 25% concentrations significantly increased E-Cadherin protein expression (P<0.01), but had no regulation effect on Alpha-catenin and beta-catenin protein expression; Feiyanning at 5% and 10% concentrations had a down-regulation effect on N-cadherin and Fibronectin protein expression (P<0.01), and had no regulation effect on vimentin protein expression.
Feiyanning play a role in inhibiting the adhesion of tumor heterogeneity and the athletic ability by regulating epithelial-mesenchymal cell marker factor expression, and thus inhibit the invasion and metastasis of lung cancer.
我们之前的体内实验（论文已发表）证实了肺炎宁对上皮细胞标志物α-连环蛋白、β-连环蛋白、E-钙黏蛋白以及间充质细胞标志物N-钙黏蛋白、纤连蛋白、波形蛋白的调控作用。基于之前的研究，本研究的目的是进一步在体外研究肺炎宁干预高转移性肺癌细胞95-D上皮-间充质细胞标志物基因及蛋白的表达情况。
体外用人高转移性肺癌95-D细胞分别给予不同浓度的肺炎宁处理，然后采用实时荧光定量PCR及蛋白质免疫印迹法检测上皮-间充质细胞标志物因子α-连环蛋白、β-连环蛋白、E-钙黏蛋白、N-钙黏蛋白、纤连蛋白及波形蛋白的mRNA及蛋白表达。
实时荧光定量PCR结果显示，与对照组相比，20%浓度的肺炎宁显著上调α-连环蛋白的表达（P<0.05），20%和25%浓度的肺炎宁显著上调E-钙黏蛋白的表达（P<0.05，P<0.01），肺炎宁在任何浓度下对β-连环蛋白均无显著影响，5%和10%浓度的肺炎宁显著下调间充质细胞标志物波形蛋白因子的表达（P<0.01），肺炎宁在任何浓度下对N-钙黏蛋白和纤连蛋白均无调控作用。蛋白质免疫印迹结果显示，5%、10%、15%、20%和25%浓度的肺炎宁显著增加E-钙黏蛋白的蛋白表达（P<0.01），但对α-连环蛋白和β-连环蛋白的蛋白表达无调控作用；5%和10%浓度的肺炎宁对N-钙黏蛋白和纤连蛋白的蛋白表达有下调作用（P<0.01），对波形蛋白的蛋白表达无调控作用。
肺炎宁可通过调控上皮-间充质细胞标志物因子表达，发挥抑制肿瘤异质性黏附及运动能力的作用，从而抑制肺癌的侵袭和转移。
