caveolin-1对喉鳞状细胞癌生物学特性的影响

Effects of caveolin-1 on biologic behavior of laryngeal squamous cell carcinoma HEp2 cell line

目的探讨细胞质膜微囊蛋白-1（caveolin-1）对喉鳞状细胞癌生物学特征的影响。方法构建caveolin-1真核表达质粒,用脂质体法转染喉鳞癌细胞株HEp2细胞,筛选出稳定高表达caveolin-1的克隆,并用实时荧光定量PCR和免疫印迹法鉴定。MMT法用于检测细胞的生长能力,软琼脂克隆形成实验用于检测细胞锚定非依赖性生长能力,流式细胞仪检测细胞周期和凋亡,免疫印迹法检测表皮生长因子受体（EGFR）和细胞外信号调节激酶（Erk1／2）的磷酸化水平,用激光共聚焦显微镜观察caveolin-1和EGFR的共定位。结果成功构建了caveolin-1真核表达质粒,转染HEp2细胞后筛选得到3株稳定高表达caveolin-1的克隆。与对照组相比,转染后的细胞生长速度明显减慢,软琼脂中克隆形成能力明显下降。流式细胞仪结果显示,高表达caveolin-1可使细胞阻滞在G0／G1期,并且促进细胞的凋亡。激光共聚焦显微镜观察显示,caveolin-1与EGFR共定位于细胞膜。免疫印迹检测显示,caveolin-1能降低EGFR和Erk1／2的磷酸化水平。结论caveolin-1能够抑制喉鳞癌细胞株HEp2的生长,并促进其凋亡;对EGFR-MAPK信号通路的负性调节可能在其抑癌机制中发挥重要作用。

Objective To investigate the effects of caveolin-1 on the biologic behavior of laryngeal squamous cell carcinoma HEp2 cell line in vitro. Methods Eukaryotic expression vector of human caveolin- 1 gene was constructed and transfected into HEp2 cells by Lipofectamine. The clones stably overexpressing caveolin-1 were identified by real-time PCR and Western blotting. Cell proliferation viability was tested by MTF assay. Anchorage-independent growth was determined by assaying colony formation in soft agar. Flow cytometry was used to assess the cell cycle and apoptosis. The relative phosphorylation level of EGFR and ERK1/2 were detected by Western blotting. Localization of caveolin-1 and EGFR were studied by laser confocal laser scanning microscopy. Results The expression vector of caveolin-1 was constructed and three clones stably overexpressing caveolin-1 were obtained. Comparing with the parental HEp2 cells, the transfected cells exhibited a slower growth rate and formed fewer colonies in soft agar. The results of FACS analysis revealed that overexpression of caveolin-1 resulted in the cell cycle arrest at G0/G1 phase and increased the apoptotic cell fraction. EGFR was found to colocalize with caveolin-1 in transfected cells by confocal laser scanning microscopy and Western blotting results showed that overexpression of caveolin-1 reduced the phosphorylation of EGFR and Erk1/2. Conclusion Overexpression of caveolin-1 suppresses the growth of HEp2 cells and induces apoptosis and inhibition of EGFR-MAPK signaling pathway may be involved in its mechanism.