摘要
目的:研究敲除E3泛素连接酶RAD18基因后,人肺腺癌A549细胞株和顺铂耐药A549(A549/DDP)细胞株FA/BRCA途径DNA修复功能的变化及其对顺铂敏感性的改变。方法:合成针对RAD18基因的siRNA(RAD18-siRNA),通过脂质体将其分别转染人A549和A549/DDP细胞株。蛋白质印迹法检测经顺铂处理的两种细胞株转染siRNA前后RAD18蛋白表达及FANCD2单泛素化水平;CCK-8法测定经顺铂处理的两种细胞株转染siRNA前后细胞增殖率的变化;免疫荧光测定胞核内FANCD2核聚小体的形成;Annexin V/PI流式细胞术测定转染前后细胞凋亡率。结果:与转染RAD18-siRNA前相比,转染后经顺铂处理的A549和A549/DDP细胞株RAD18表达均明显降低;FANCD2蛋白单泛素化水平下调和核聚小体形成减少;同时细胞增殖率明显降低,细胞凋亡率则明显增加。结论:应用RNA干扰技术敲除RAD18基因可通过抑制A549和A549/DDP细胞株FA/BRCA途径的DNA损伤修复功能,增加这两种细胞株对顺铂的敏感性。
Objective: To investigate the changes of the FA/BRCA pathway DNA repair function,as well as the sensitivity to cisplatin in human lung adenocarcinoma A549 cells and A549 / DDP cell lines( a cisplatin-resistance A549 cell line) after knockdown of the E3 ubiquitin ligase RAD18 gene by siRNA technique. Methods: Using RNA interfering approach to transfect RAD18-siRNA into A549 and A549 / DDP cells by Lipofectamine. Western blotting was carried out to test the expression of RAD 18 protein,and the FANCD2 monoubiquitination levels,as indicated by the ratio of FANCD2-L( long form of FANCD2) to FANCD2-S( short form of FANCD2) in the two cells treated with cisplatin before and after RAD18-siRNA transfection. CCK-8 technique was used to detect the cell proliferation rate of the two cells treated with cisplatin pre-and-post transfection of RAD18-siRNA. Immunofluorescence assay was performed to inspect the formation of FANCD2 nuclear foci. Flow cytometry using Annexin V / PI methods was conducted to measure cell apoptosis rate. Results: After transfection of RAD18-siRNA,the expression of RAD18 protein,and the levels of FANCD2 monoubiquitination( L / S ratio),and the nuclear foci formation of FANCD2 had a significant decline in A549 and A549 / DDP cells treated with cisplatin compared with before transfection. Meanwhile,we found that knockdown of RAD 18 gene can suppress the proliferation rate and promote apoptosis induced by cisplatin in the two cells( both P < 0. 05). Conclusion: Knockdown of RAD18 gene by RNA interfering approach can potentiate the sensitivity to cisplatin in A549 and A549 / DDP cells through inhibition of FA / BRCA pathway DNA damage repair function.
出处
《江苏大学学报（医学版）》
CAS
2015年第2期103-108,113,共7页
Journal of Jiangsu University：Medicine Edition
