中国癌症杂志 ›› 2017, Vol. 27 ›› Issue (7): 527-532.doi: 10.19401/j.cnki.1007-3639.2017.07.002

• 论著 • 上一篇    下一篇

跨膜接头蛋白CBP对皮肤鳞癌细胞A431生长增殖的负调控作用研究

李美佳1,张维娜1,渠开攀1,王 冰2   

  1. 1. 青岛大学附属医院烧伤整形外科,山东 青岛 266000 ;
    2. 青岛大学医学院免疫教研室,山东 青岛 266000
  • 出版日期:2017-07-30 发布日期:2017-08-16
  • 通信作者: 王 冰 E-mail: wangbing@qdu.edu.cn

CBP mediates negative regulation of growth and proliferation of cutaneous squamous cell carcinoma A431 in vitro

LI Meijia1, ZHANG Weina1, QU Kaipan1, WANG Bing2   

  1. 1. Department of Burn and Plastic Surgery, the Affiliated Hospital of Qingdao University, Qingdao 266000, Shandong Province, China; 2. Department of Immunology, Medical College of Qingdao University, Qingdao 266000, Shandong Province, China
  • Online:2017-07-30 Published:2017-08-16
  • Contact: WANG Bing E-mail: wangbing@qdu.edu.cn

摘要: 背景与目的:跨膜接头蛋白(Csk-binding protein,CBP)是新发现的Src家族成员,与多种肿瘤的发生有关。该研究旨在观察CBP基因过表达对皮肤鳞癌细胞系A431增殖及细胞凋亡的影响,探讨其相关的分子机制。方法:构建CBP-增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)融合蛋白的慢病毒过表达载体,采用反转录病毒转染的方法建立CBP过表达的A431细胞株。实验分为亲本细胞组(未进行基因转染的A431细胞)、对照组(A431细胞转染仅含EGFP阴性对照病毒)和实验组(A431细胞转染CBP-EGFP病毒)。在激光共聚焦显微镜下观察细胞转染率,验证转染成功与否;CCK-8法检测CBP过表达对A431细胞增殖能力的影响,并采用流式细胞术(flow cytometry,FCM)检测对细胞凋亡的影响;实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)和蛋白[质]印迹法(Western blot)检测Lck、Csk和Fyn三种上游信号转导分子分别在mRNA和蛋白中的表达水平变化。结果:建立了稳定过表达CBP的A431细胞株;CCK-8法结果提示,CBP过表达明显抑制细胞生长,第2~6天的组间差异有统计学意义(P<0.05);FCM检测显示,实验组细胞凋亡率显著增加,与亲本细胞组及对照组相比差异有统计学意义(P<0.001);RTFQ-PCR结果显示,实验组A431细胞Lck mRNA的相对表达水平显著下调(P<0.001),实验组细胞Csk和Fyn mRNA的表达分别约为亲本细胞组的1.6倍和3.8倍,表达显著上调(P<0.001);Western blot结果表明,实验组Lck蛋白的相对表达水平明显下降(P<0.001),实验组细胞Csk和Fyn蛋白与亲本细胞组和对照组相比表达明显增加(P<0.001)。结论:CBP过表达可抑制皮肤鳞癌细胞增殖,诱导细胞凋亡及坏死。CBP通过调节上游信号转导通路中的蛋白酪氨酸激酶Lck、Csk和Fyn来调控细胞的增殖活性。

关键词: 皮肤鳞癌, 跨膜接头蛋白, 细胞增殖, 细胞凋亡

Abstract: Background and purpose: The transmembrane adaptor Csk-binding protein (CBP), a recently identified tyrosine kinase of the Src family, is implicated in various aspects of cancer cell biology. This study aimed to investigate the effect of the transmembrane protein CBP on the proliferation and apoptosis ability of A431 cells of human cutaneous squamous cell carcinoma and the implicated molecular mechanism. Methods: The CBP-EGFP lentiviral vector was constructed. A431 cells were transfected with CBP-EGFP lentiviral vector or negative-EGFP lentiviral vector, or remained untransfected. The transfection efficiency was detected by laser confocal microscope. After additional culture, the proliferation potential of A431 cells was assayed with CCK-8, and the apoptotic rate was assessed by flow cytometry (FCM). Lck, Csk and Fyn mRNA levels were detected by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and their protein levels by Western blot. Results: The A431 cell line with overexpression of PAG was constructed successfully. CCK-8 results suggested that overexpression of CBP markedly inhibited cell growth, with statistically significant differences at 2-6 days between the groups (P<0.05). FCM showed that both the apoptotic rate and the death rate of the cells transfected with CBP-EGFP lentiviral vector were increased significantly compared to those of the cells transfected with negative-EGFP lentiviral vector or untransfected cells (P<0.001). RTFQ-PCR results showed that the Lck mRNA relative expression level of the cells transfected with CBP-EGFP lentiviral vector was significantly reduced (P<0.001), but Csk and Fyn mRNA expression levels were 1.6 times and 3.8 times as high as the untransfected cells, respectively (P<0.001). Western blot results showed that the Lck protein level was significantly decreased after transfection with CBP-EGFP lentiviral vector (P<0.001), whereas the Csk and Fyn protein levels were significantly increased (P<0.001). Conclusion: The ectopic expression of CBP might inhibit the proliferation or growth of A431 cells, induce cell apoptosis and accelerate cell death, which may be related to down-regulation of Lck and up-regulation of Csk and Fyn expression.

Key words: cutaneous squamous cell carcinoma, Csk-binding protein, proliferation, apoptosis