黄芩素对A431细胞埃兹蛋白表达及侵袭力的影响

Effects of baicalein on the expression of ezrin protein in and invasiveness of a skin squamous cell carcinoma cell line A431

目的探讨黄芩素是否通过抑制埃兹蛋白来实现抑制A431细胞增殖、细胞周期进程及伪足的形成。方法采用细胞划痕、微孔滤膜法（Transwell）检测黄芩素对A431细胞爬行的影响；RT—PCR检测黄芩素对A431细胞埃兹蛋白mRNA表达量的影响；Western印迹检测黄芩素及siRNA对A431细胞埃兹蛋白表达及其磷酸化的影响；扫描电镜观察黄芩素及siRNA对A431细胞伪足形成的影响。结果细胞划痕实验显示，培养24h时，黄芩素5、10、20p,mol／L组细胞闭合宽度占原宽度的比例分别为13．3％±1．7％、7．6％±1．6％和5．9％±1．3％，黄芩素呈浓度依赖性抑制A431细胞爬行运动，黄芩素各组与阴性对照组（16．3％±2．3％）比较，差异均有统计学意义。Transwell实验证实，5、10、20μmol／L黄芩素处理A431细胞48h后，穿过人工基底膜的细胞数量分别为（46．5±3．8）、（34．3±3．4）和（25.3±2．3）个／Hp，各处理组与阴性对照组（56．3±3.8个／Hp）经方差分析，P〈0．01；而与siRNA组（28.3±2．1个/Hp）比较，P〉0．05。Western印迹及RT—PCR检测显示，0、5、10、20、40μmol／L黄芩素处理A431细胞48h后，黄芩素呈浓度依赖性地抑制A431细胞埃兹蛋白膦酸化埃兹蛋白及埃兹蛋白mRNA表达，埃兹蛋白mRNA与β-肌动蛋白mRNA灰度值之比值与阴性对照组比较，P值均〈0．01；而与siRNA组比较，P〉0．05；埃兹蛋白、磷酸化埃兹蛋自在A431细胞中的表达量（与β-肌动蛋白灰度值之比）与阴性对照组比较，P值均〈0．01：而40la,mol／L黄芩素处理组与siRNA处理组比较，P〉0．05。扫描电镜显示，48h后20μmol/L黄芩素组A431细胞伪足数量为（5．3±1．9）个，长度明显缩短，与阴性对照组（22．6±2．8个）比较，P〈0．01；siRNA组为（4．5±2．8）个．与阴性对照组比较，P〉0．05。结论黄芩素可能通过直接或间接抑制埃兹蛋白表达及其磷酸化而抑制A431细胞的增殖、爬行，从而达到抗肿瘤增殖及浸润转移的目的。

Objective To investigate whether baicalein inhibits the proliferation, cell cycle of and pseudopod formation in A431, a skin squamous cell carcinoma eel/line, by suppressing the expression of ezrin protein. Methods A431 cells were grouped to be transfected with ezrin-targeting siRNA （siRNA group）, treated with baicalein of 5, 10, 20, 40 μmol/L, respectively （baicalein group）, or remain untreated （control group）. After additional culture, wound healing assay and Transwell assay were performed to observe the migration and invasion of A431 cells, RT-PCR to detect the mRNA expression of ezrin in A431 cells, Western blot and immunofluorescenee to measure the expression of ezrin protein and its phosphorylation. The pseudopod formation in A431 cells was observed by using scanning electron microscopy. Results After 24-hour culture, wound healing assay displayed that the percent wound closure was 13.3 ± 1.7, 7.6 ± 1.6 and 5.9 ± 1.3, respectively, in A431 cells treated with baicalein of 5, 10, 20 μmol/L, significantly lower than that in untreated A431 cells （16.3 ± 2.3, all P 〈 0.01 ）, and the inhibition of baicalein on the migration of A431 cells was concentration-dependent. In the Transwell assay, a significant decrease was observed in the number of A431 cells per high power field permeating through the artificial basement membrane in the groups treated with baica/ein of 5, 10, 20 μmol/L for 48 hours compared with the control group （46.5 ± 3.8, 34.3 ± 3.4, 25.3 ± 2.3 vs 56.3 ± 3.8, all P 〈 0.01 ）, whereas no significant difference was noted between these baicalein-treated groups and siRNA-transfeeted group （28.3 ±2.1, all P 〉 0.05）. RT-PCR analysis showed that the mRNA expression of ezrin in baicaleintreated A431 cells significantly decreased compared with that in untreated cells （all P 〈 0.01 ）, but showed no difference from that in siRNA group （ P 〉 0.05 ）. A statistical difference was also observed in the expression of ezrin and phosphorylated ezrin protein between baicalein-treated A431 cells and untreated cells （all P 〈 0.05 ）, but not between 40 Ixmol/L baicalein-treated A431 cells and siRNA-transfected cells （P 〉 0.05）. Furthermore, the suppression of baicalein on ezrin protein and mRNA expression was concentration dependent. The number of pseudopod per cell was significantly lower in 20 μmol/L baicalein-treated A431 cells and siRNA-transfected cells than that in untreated A431 cells （5.3 ± 1.9, 4.5 ± 2.8 vs 22.6 ± 2.8, both P〈 0.01）, while no significant difference was observed between the former two groups of cells （P 〉 0.05）; the length of pseudopodia also reduced in baicalein-treated cells. Conclusions Baicalein may inhibit the proliferation and invasion of A431 cells by directly or indirectly suppressing the expression of ezrin and phosphorylated ezrin, which in turn contributes to the effect of baicalein against tumor proliferation and metastasis.