FAAP20和RAD51C基因敲减对肺癌细胞顺铂敏感性的影响  

Impact of FAAP20 and RAD51C gene knockdown by siRNA on the sensitivity of lung cancer cells to cisplatin

作　　者：陈康[1] 李坚[1] 李玫瑜 CHEN Kang LI Jian LI Mei-yu(Department of Respiratory Medicine, Affiliated Hospital of Jiangsu University, Zhenjiang Jiangsu 212001, China)

机构地区：[1]江苏大学附属医院呼吸内科,江苏镇江212001

出　　处：《江苏大学学报（医学版）》2016年第1期5-10,16,共7页Journal of Jiangsu University：Medicine Edition

摘　　要：目的:研究敲减范可尼贫血(FA)通路上游FAAP20和下游RAD51C基因对人肺癌Calu-1细胞株顺铂敏感性的影响。方法:合成针对FAAP20和RAD51C基因的si RNA(FAAP20-si RNA和RAD51C-si RNA),用脂质体转染试剂将他们分别和同时转染于人肺癌Calu-1细胞株。蛋白质印迹法检测si RNAs转染后细胞FAAP20和RAD51C蛋白的表达以验证转染效率,并检测FANCD2蛋白单泛素化水平;CCK-8法测定转染前后的细胞增殖率;Annexin V/PI流式细胞术测定转染前后细胞凋亡率;免疫荧光法观察转染前后细胞核内FANCD2蛋白核聚小体的形成。结果:与转染前比较,转染FAAP20-si RNA和RAD51C-si RNA后经顺铂处理的Calu-1细胞FAAP20和RAD51C蛋白表达均明显降低,表明转染有效。敲减FAAP20基因后顺铂诱导的FANCD2蛋白单泛素化水平降低,核聚小体形成减少,而RAD51C基因敲减对FANCD2蛋白单泛素化和核聚小体形成并无明显影响。敲减FAAP20或RAD51C基因均可增强顺铂诱导的细胞增殖抑制和细胞凋亡作用,同时共敲减这两个基因则进一步增强顺铂对Calu-1细胞的杀瘤效应。结论:敲减FAAP20和RAD51C基因可增加Calu-1细胞对顺铂的敏感性,FAAP20和RAD51C基因的DNA损伤修复功能并不完全作用在同一条路径(范可尼贫血通路)。Objective： To investigate the effect of RNA interference （RNAi） targeting Fanconi anemia pathway FAAP20 and RAD51C genes on the sensitivity of lung cancer cells to cisplatin. Methods： Small interference RNAs targeting FAAP20 and RAD51 C genes（ FAAP20-siRNA and RAD51C-siRNA） were trans- fected into Calu-1 cells by lipofectamine 2000. Western blotting was used to verify siRNA transfection effi- cacy, and determine the FANCD2 monoubiquitination levels induced by cisplatin, which were indicated by the radio of monoubiquitination-FANCD2 （L） and non-monoubiquitination-FANCD2 （ S ）, after and before transfection. CCK-8 assay was carried out to detect the cell proliferation rate of Calu-1 cells treated with cis- platin pre-transfection and post-transfection. Flow cytometry using Annexin V/PI methods was performed to measure cell apoptosis. Immunofluorescence stain had been done to test the formation of FANCD2 nuclear foci. Results： Knockdown of FAAP20 gene significantly inhibited FANCD2 monoubiquitination and nuclear foci formation produced by cisplatin in Calu-1 cells. However, RAD51C gene knockdown did not influence FANCD2 monoubiquitination and nuclear foci formation. The effects of cell proliferation suppression and ap- optosis induced by cisplatin were markedly enhanced by individually depleting FAAP20 and RAD51 C gene. Furthermore, double depletion of FAAP20 and RAD51 C genes further increased the cytotoxicity of cisplatin to Calu-1 cells. Conclusion： Depletion of FAAP20 and RAD51C gene by siRNA can potentiate the sensitiv- ity of lung cancer cells to cisplatin through inhibition of Fanconi anemia pathway. The sensitization effect tocisplatin was further increased by co-depletion of the two genes, suggesting that FAAP20 and RAD51C do not completely function in a common pathway for the repair of DNA damage.

关 键 词：FAAP20 RAD51C FANCD2 SIRNA 顺铂 肺癌细胞 

分 类 号：R730.54[医药卫生—肿瘤]