共抑制RAD18和REV1基因显著增加顺铂对肺癌细胞的毒性

目的: 探讨采用基因敲减技术抑制跨损伤合成（translesion synthesis,TLS）通路和范可尼贫血（Fanconi anemia,FA)通路上游的RAD18基因与TLS通路的REV1基因后增加顺铂对人肺腺癌耐药细胞株（A549/DDP）细胞毒性的可行性及其机制。方法: 蛋白质印迹法测定顺铂诱导的A549和A549/DDP细胞RAD18和REV1蛋白表达；应用脂质体转染试剂转染针对RAD18基因的siRNAs (RAD18 siRNA)，检测A549/DDP细胞转染前后顺铂诱导的FANCD2和PCNA蛋白单泛素化水平；CCK 8法检测分别转染RAD18 siRNA、REV1 siRNA以及两者共转染前后经顺铂处理的A549/DDP细胞的增殖率；流式细胞术检测转染前后A549/DDP细胞凋亡率和细胞周期。结果： A549/DDP细胞转染RAD18 siRNA和REV1 siRNA后，RAD18和REV1蛋白表达明显降低，表明转染有效，基因敲减成功。转染RAD18 siRNA后顺铂诱导的FANCD2和PCNA蛋白的单泛素化水平明显下降，提示FA通路和TLS通路的激活受到抑制。分别转染RAD18 siRNA或REV1 siRNA均可增强顺铂对A549/DDP的细胞毒性，增加顺铂诱导的细胞凋亡率，并增强细胞周期S/G2期的阻滞效应。共转染RAD18 siRNA和REV1 siRNA后，顺铂对A549/DDP细胞的毒性增强作用较单转染更为显著，顺铂诱导的细胞凋亡进一步增加。 结论： 敲减A549/DDP细胞的RAD18和REV1基因可通过抑制FA通路和TLS通路的DNA损伤修复功能，增加A549/DDP细胞对顺铂的敏感性；RAD18和REV1基因共敲减对顺铂的这一增敏作用更加明显，显示出A549/DDP细胞对顺铂耐药的逆转效应。

Co-inhibition of RAD18 and REV1 gene markedly enhance the cytotoxicity of cisplatin to lung cancer A549 /DDP cell line

Objective: To explore the effects of RAD18 and REV1 gene knockdowns on cisplatin cytotoxicity in cisplatin resistant A549/DDP cell line. Methods: The expression of RAD18 and REV1 protein, and the FANCD2 and PCNA monoubiquitination level were detected by Western blotting. The siRNAs against RAD18 and REV1 genes(RAD18 siRNA and REV1 siRNA) were transfected into A549/DDP cells using Lipofectamine. The CCK 8 assay was used to detect the changes of cell proliferation rate following treatment with cisplatin before and after transfection of RAD18 siRNA and REV1 siRNA in A549/DDP cells. Cisplatin induced cell apoptosis and cell cycle were detected by flow cytometry in A549/DDP cells pre and post transfection of RAD18 siRNA and REV1 siRNA. Results: The expressions of RAD18 and REV1 proteins were significantly decreased after transfection of RAD18 siRNA or REV1 siRNA, indicating that the transfections were successful. Cisplatin induced monoubiquitinations of FANCD2 and PCNA were significantly decreased after transfection with RAD18 siRNA, suggesting that the activation of the FA pathway and the TLS pathway was suppressed. Individual knockdown of RAD18 or REV1 by siRNAs transfection enhanced the cytotoxicity of cisplatin to A549/DDP cells, promoted cell apoptosis induced by cisplatin, and resulted in the arrest of S/G2 phase. Co knockdown of RAD18 and REV1 genes further enhanced the cytotoxicity of cisplatin to A549/DDP cells compared to RAD18 or REV1 knockdown alone. Conclusion: Knockdowns of RAD18 gene of the TLS pathway and the Fanconi anemia（FA） pathway upstream and REV1 gene of the TLS pathway could increase the sensitivity of cisplatin resistant A549/DDP cells to cisplatin by inhibiting the DNA damage repair capacity,and the sensitization can be further potentiated by co knockdown of RAD18 and REV1.