柠檬酚对人肝癌HepG2细胞增殖、迁移和骨架相关蛋白表达的影响及其与蛋白作用方式研究

Study on the Effects of Citrusinol on the Proliferation,Migration and the Expression of Skeleton-related Proteins of Human Hepatocellular Cells HepG2 and Its Interaction Mode with Skeleton-related Proteins

目的:研究柠檬酚对人肝癌HepG2细胞增殖、迁移及骨架相关蛋白表达的影响,并探讨其与骨架相关蛋白的相互作用方式。方法:采用CCK-8法检测不同浓度柠檬酚(12.5、25、50、100、200μmol/L)作用24 h后对HepG2细胞增殖的影响。将HepG2细胞分为阴性对照组和柠檬酚低、高浓度组(50、100μmol/L柠檬酚),加入相应药物作用24 h后,采用划痕试验检测细胞的迁移能力并计算细胞迁移率,采用逆转录-聚合酶链式反应和Western blotting法检测细胞中聚合纤维状肌动蛋白(F-actin)、β-微管蛋白(β-tubulin)及埃兹蛋白(Ezrin)的mRNA和蛋白表达水平。利用分子对接软件Schrodinger 2015分析柠檬酚与上述3种蛋白的分子作用方式。结果:柠檬酚对HepG2细胞增殖具有显著抑制作用(P<0.05或P<0.01),且呈剂量依赖趋势。与阴性对照组比较,柠檬酚低、高浓度组细胞迁移率和F-actin、β-tubulin、Ezrin的mRNA及蛋白表达水平显著降低(P<0.05或P<0.01)。分子对接结果显示,柠檬酚可与上述3种细胞骨架蛋白形成氢键和疏水键。结论:柠檬酚可抑制肝癌HepG2细胞的增殖和迁移;其作用机制可能与下调F-actin、β-tubulin、Ezrin的m RNA及蛋白表达有关;其与骨架相关蛋白的作用方式可能是形成氢键或疏水键。

OBJECTIVE:To study the effects of citrusinol on proliferation,migration and the expression of skeleton-related proteins of human hepatocellular cells HepG2,and to investigate its interaction mode with skeleton-related proteins. METHODS:CCK-8 assay was used to detect the effects of different concentrations(12.5,25,50,100,200 μ mol/L)of citrusinol on the proliferation of HepG2 cells for 24 h. HepG2 cells were divided into negative control group,citrusinol low-concentration and high-concentration groups(50,100 μmol/L citrusinol). After treated for 24 h,the migration of HepG2 cells was detected by cell scratch test;cell migration rate was calculated. mRNA and protein expression of F-actin,β-tubulin and Ezrin in HepG2 cells were determined by RT-PCR and Western blotting assay. Molecular docking software Schrodinger 2015 was used to analyze the interaction mode between citrusinol and above 3 kinds of proteins. RESULTS:Citrusinol showed significant inhibition effect on the proliferation of HepG2 cells(P<0.05 or P<0.01), in dose-dependent trend. Compared with negative control group, cell migration, mRNA and protein expression levels of F-actin, β-tubulin, Ezrin were decreased significantly in citrusinol low-concentration and high-concentration groups(P<0.05 or P<0.01). Molecular docking results showed that the citrusinol could form hydrogen bond and hydrophobic bond with the above 3 skeleton-related proteins. CONCLUSIONS:Citrusinol can inhibit the proliferation and migration of HepG2 cells,the mechanism may be associated with the down-regulation of mRNA and protein expression of F-actin,β-tubulin and Ezrin. The mode of its interaction with skeleton-related proteins may be the formation of hydrogen bond or hydrophobic bond.