Co-inhibition Of RAD18 And REV1 Markedly Enhance The Cytotoxicity Of Cisplatin To Lung Cancer A549/DDP Cell Line

ObjectiveTo investigate the effects of single or co-inhibition of RAD18 and REV1 genes in the Fanconi anemia（FA）and translesion synthesis（TLS）pathways on the sensitivity of cisplatin in human lung adenocarcinoma cisplatin-resistant cell line（A549/DDP）,and probe the related mechanism.MethodsWestern blotting was used to determine the expression levels of RAD18 and REV1 protein in human lung adenocarcinoma cell lines（A549）and A549/DDP cells upon DDP treatment.The siRNAs targeting RAD18 and REV1（RAD18-siRNA and REV1-siRNA）were transfected into A549/DDP cells respectively by liposome transfection reagent.The effectiveness of transfection was verified by Western blot assay.The cisplatin-induced by monoubiquitination levels of FANCD2（ratio of the FANCD2 fragment length,that FANCD2-L/S ratio）and PCNA were detected using Western blotting after silencing of RAD18 gene.The CCK-8 assay was carried out to determine the proliferation rates of A549/DDP cells treated with cisplatin for 24 hours and 48 hours before and after individual-or co-transfection of RAD18-siRNA and REV1-siRNA.The Flow cytometry was conducted to analyze the apoptosis rate and cell cycle of A549/DDP cells treated with 10μg/m L cisplatin for 48h before and after individual-and co-transfection of RAD18-siRNA and REV1-siRNA.Results（1）Upon treatment with different concentrations of cisplatin,the expression levels of RAD18 and REV1 proteins in A549/DDP cells were significantly increased compared to those in A549 cells in a concentration-dependent manner（all P<0.05）,suggesting that the elevated expressions of RAD18 and REV1 proteins are involved with cisplatin resistance in A549/DDP cells.（2）After transfection with RAD18-siRNA and REV1-siRNA,western blotting showed markedly reduced levels of RAD18 and REV1 proteins in A549/DDP cells（P<0.05）,verifying that the transfections were effective and the two genes were successfully silenced.（3）The FANCD2 and PCNA monoubiquitination levels induced by different concentrations of cisplatin were dramatically decreased following RAD18-siRNA transfection（P<0.05）,which indicated that the activation of the FA and TLS pathways were inhibited.This data suggest that RAD18 gene acted in the upstream of the FA pathway and concurrently activate both the FA pathway and TLS pathways in response to cisplatin.（4）The half maximal inhibitory concentrations(IC₅₀)of cisplatin in A549/DDP cells were significantly reduced after transfection with RAD18-siRNA or REV1-siRNA as compared with those before transfections with the siRNAs（P<0.05）.The results indicate that silencing of RAD18 or REV1 enhanced cisplatin cytotoxicity to A549/DDP cells.Moreover,double-silencing of RAD18 and REV1 gene further potentiated the killing effect of cisplatin on A549/DDP cells（all P<0.05）.（5）Flow cytometry showed that the percentage of apoptotic cells induced by cisplatin（10μg/m L）was significantly elevated in A549/DDP cells after silencing of RAD18 or REV1.Furthermore,double-silencing with RAD18 and REV1 notably increased the rates of apoptotic cells induced by cisplatin compared to single transfection with either the siRNA（P<0.05）.Cell cycle analysis found that the proportion of cell in G₂phase was markedly augmented in A549/DDP cells post-transfection with either the siRNA（all P<0.05）.Likewise,double-transfection with the two siRNAs resulted in further increase of cells in G₂ phase,indicating that silencing of RAD18 and REV1lead to the arrest of cells in G₂ phase and stalled DNA replication（all P<0.05）.ConclusionHigh expressions of RAD18 and REV1 in the FA and TLS pathways are involved in ciplatin resistance in A549/DDP cells.Knockdown of RAD18 or REV1genes can enhance the sensitivity of A549/DDP cells to cisplatin through inhibition of DNA damage repair executed by the FA and TLS pathways.The sensitization of A549/DDP cells to cisplatin can be further potentiated by double-knockdown of RAD18 and REV1 genes,indicating that co-knocdown of REV1 and RAD18 gene could reverse resistance of A549/DDP cells to cisplatin.