DEPDC1稳定沉默CNE-1细胞株的构建与分析

Establishment and analysis of nasopharyngeal carcinoma cells CNE-1 with DEPDC1 stable silencing

摘要:

目的：为了进一步探讨DEPDC1（DEP domain containing 1）在肿瘤发生发展中的作用。DEPDC1是近年发现的一个肿瘤相关基因，前期研究结果显示其在鼻咽癌细胞周期及癌变过程中具有作用。方法：利用qRT-PCR和免疫组织化学染色分别检测DEPDC1在18例新鲜样品和44例组织芯片肿瘤患者中的表达情况。人鼻咽癌细胞系CNE-1稳定沉默DEPDC1后，流式细胞检测细胞增殖并利用Transwell小室分析侵袭迁移情况。结果： DEPDC1在鼻咽癌组织中表达量（0.699±0.521）明显高于正常组织（0.408±0.183），差异有统计学意义（P＜0.05）。DEPDC1稳定沉默后，CCK8检测结果表明 CNE-1细胞增殖速度减慢。流式细胞检测的结果显示，G1期细胞减少，G2/M期细胞增多，细胞周期进程受阻。划痕及Transwell实验结果表明，DEPDC1稳定沉默明显减弱了CNE-1细胞运动，侵袭及迁移能力（77.033±5.183 vs. 20.137±2.828， 107.336±5.033 vs. 26.326±1.414），差异均有统计学意义（P＜0.05）。进一步定量qRT-PCR及Western blot检测发现，DEPDC1稳定沉默导致上皮细胞标志分子E-cad－herin（0.457±0.022）显著上调、而间质细胞标志分子Vimentin（0.780±0.063）、N-cadherin（0.780±0.063）以及EMT上游关键转录因子Twist1（0.710±0.034）下调（P＜0.05）。结论：本研究成功构建了DEPDC1稳定沉默细胞株，与前期siRNA介导的瞬时沉默相似的是，发现DEPDC1稳定沉默可抑制鼻咽癌细胞的增殖、侵袭迁移，并改变EMT关键分子的表达，为进一步探索DEPDC1在鼻咽癌中的作用机制奠定了基础。

Abstract:

Objective：To investigate the role of DEP domain containing 1（DEPDC1） in the pathogenesis of nasopharyngeal carcinoma. Methods：The expression level of DEPDC1 in archival tissue samples from nasopharyngeal carcinoma patients and healthy individuals was examined by quatitative RT-PCR and immunohistochemical assay. The nasopharyngeal carcinoma cell line CNE-1 with stable suppression of DEPDC1 expression was established by short hairpin RNA，and their proliferative capability and cell cycle status were separately assessed by CCK-8 assay and FACS assay. Additionally，their migratory and invasive abilities were detected using wound healing assay，Transwell migration and invasion assays. Results：The expression level of DEPDC1 in the tumor tissues（0.699±0.521） was significantly higher than that in the normal tissues（0.408±0.183）（P＜0.05）. Suppression of DEPDC1 in CNE-1 cells revealed significant inhibition of cellular proliferation as well as G2/M phase arrest. Depletion of DEPDC1 also remarkably attenuated cell motility and invasiveness accompanied by the reduction of Twist1（0.710±0.034） and Vimentin （0.780±0.063）（P＜0.05）. Conclusion：Our findings uncover a novel role for DEPDC1 in cell cycle progression and metastasis of nasopharyngeal carcinoma，and provide a rationale for its potential in the diagnosis and/or therapy of nasopharyngeal carcinoma.