抑制肺鳞癌细胞FANCM和USP1基因对顺铂的增敏效应

Effect of sensitization to cisplatin by inhibiting FANCM and USP1 in lung squamous carcinoma cells

目的:研究抑制FANCM和USP1基因后,人肺鳞癌SK-MES-1细胞株FA/BRCA通路激活状态以及对顺铂敏感性的变化。方法:用脂质体转染试剂将FANCM-siRNA和USP1-siRNA分别和共转染于SK-MES-1细胞株,采用蛋白质印迹法测定转染后FANCM和USP1蛋白表达,并检测转染前后经DDP处理后细胞FANCD2蛋白单泛素化水平。免疫荧光染色检测胞核内FANCD2核聚小体表达。CCK-8法测定转染前后细胞生存率的变化;Annexin V/PI流式细胞术测定转染前后细胞凋亡率。结果:转染FANCM-siRNA和USP1-siRNA后,SK-MES-1细胞中FANCM和USP1蛋白表达均较转染前明显降低。转染FANCM-siRNA后经顺铂诱导的FANCD2单泛素化水平和核聚小体表达明显下调;转染USP1-siRNA后,顺铂诱导的FANCD2单泛素化水平和核聚小体形成明显增加;转染USP1-siRNA后细胞生存率和细胞凋亡率高于转染FANCM-siRNA,同时共转染USP1-siRNA+FANCM-siRNA后细胞对顺铂的致敏效应与转染USP1-siRNA相似。结论:沉默FANCM和USP1基因后,通过阻断FA/BRCA通路,抑制其DNA修复功能,导致SKMES-1细胞对顺铂的敏感性增高,其中沉默USP1基因的增敏效应更为明显。

Objective ： To explore the effect of inhibition of FA/BRCA pathway upstream genes FANCM and USP1 on the sensitivity of lung squamous cell carcinoma SK-MES-1 to cisplatin. Methods： FANCM- siRNA and USPl-siRNA were transfected into SK-MES-1 cells using RNA interference （RNAi） technique according to manufacture＇s instructions. The transfection efficacy was verified by testing the FANCM and USP1 protein expression using Western blotting, which was also used to detect the levels of FANCD2 monoubiquitination. CCK-8 technique was conducted to detect the survival of SK-MES-1 cells treated with cisplatin pre-transfection and post-transfection of USPI-siRNA and FANCM-siRNA. Flow cytometry using Annexin V/PI methods was performed to measure cell apoptosis. Immunofluorescence assay was done to de- termine the expression of FANCD2 nuclear loci. Results ： FANCM and USP1 proteins expressions were ob- viously decreased after transfection of FANCM-siRNA and USP1-siRNA compared with before transfection. FANCM gene silence resulted in down-regulation of the FANCD2 monoubiquitination and nuclear loci ex- pression. The USP1 silence increased the FANCD2 monoubiquitination levels and nuclear foci expression. Meanwhile, the silencing of the two genes significantly suppressed the cell survival and promoted cell apop- tosis induced by cisplatin. The sensitizing effect to cisplatin in silencing USP1 was more obvious than in si- lencing FANCM. Conclusion ： Depletion of FANCM and USP1 gene by RNAi can enhance the sensitivity of lung squamous carcinoma cells to cisplatin through depression of the FA/BRCA pathway DNA repair func- tion.