沉默FANCD2对人头颈鳞癌SIHN-005A细胞化疗敏感度的影响及其机制

包奕琳; 李晋; 赵飞鹏; 梁灼萍; 徐伟; 覃纲

doi:10.3971/j.issn.1000-8578.2017.03.002

沉默FANCD2对人头颈鳞癌SIHN-005A细胞化疗敏感度的影响及其机制

Effects and Mechanisms of FANCD2 Knock Down on Chemotherapy Sensitivity of Head and Neck Squamous Cell Carcinoma Cells SIHN-005A

摘要

摘要:
目的研究FANCD2 shRNA干扰对人头颈鳞癌（head and neck squamous cell carcinoma, HNSCC）细胞SIHN-005A化疗敏感度的影响，并初步探讨其可能机制。
方法应用前期实验获得的FANCD2基因沉默效应稳定的人HNSCC SIHN-005A细胞，以嘌呤霉素持续筛选；Western blot检测沉默效应；CCK-8法检测顺铂作用后细胞增殖抑制率；Hoechst法检测细胞凋亡；Western blot检测Cyclin D1、Bax/Bcl-2、HIF-1α蛋白的表达。
结果实验组细胞FANCD2蛋白表达明显下降，FANCD2蛋白沉默效率达60.5%。实验组细胞增殖抑制率明显高于阴性对照组和空白对照组，且具有浓度及时间依赖性，实验组细胞对CDDP的IC₅₀值明显低于阴性对照组和空白对照组。FANCD2基因沉默后实验组细胞凋亡率明显增高。加用CDDP后，Cyclin D1、Bcl-2、HIF-1α蛋白表达水平较加用CDDP前明显下调，Bax蛋白明显增强。
结论沉默FANCD2可增加SIHN-005A细胞对化疗药物CDDP的敏感度，其机制可能与FANCD2 shRNA干扰引起Bax/bcl-2、Cyclin D1、HIF-1α的表达变化有关。

Abstract:
Objective To investigate the effects of shRNA-mediated FANCD2 knock down on the chemotherapy sensitivity to CDDP of human head and neck squamous cell carcinoma (HNSCC) cells SIHN-005A and the underlying mechanisms.
Methods The human HNSCC cells SIHN-005A which FANCD2 gene was stably silenced were obtained from our previous experiments and puromycin was used as the selection agent. The silencing effect was detected by Western blot. CCK-8 assay was used to observe the inhibition ratio of cells growth. The apoptosis and morphology changes were detected and observed by fluorescent Hoechst staining. The cell cycle D1 protein (Cyclin D1), Bax, Bcl-2 and HIF-1α were detected by Western blot.
Results The FANCD2 protein expression was significantly decreased in experimental group (shRNA) and the silencing efficiency reached 60.5%. The proliferation inhibition rate was significantly higher in the experimental group (shRNA) than those in the negative control group (shRNA-C) and blank control group (Control), and it was in a concentration and time-dependent manner. IC₅₀ of CDDP concentration of shRNA group was much lower than both shRNA-C group and Control group. The apoptosis rate was increased significantly in shRNA group. Cyclin D1, Bcl-2 and HIF-1α protein expression were significantly down-regulated in experimental group after CDDP administration, the expression of Bax protein was significantly increased in the experimental group.
Conclusion shRNA-mediated FANCD2 knock down could improve the sensitivity of HNSCC SIHN-005A cells to CDDP. The mechanism may be related to the changes in the protein expression of Bax/Bcl-2, Cyclin D1 and HIF-1α which were caused by FANCD2 knock down.

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