Effect Of Sensitization To Cisplatin By Inhibiting FA/BRCA Pathway Upstream Genes In Lung Squamous Carcinoma Cells

Objective: FA/BRCA pathway plays a critical role in repir of DNA damage induced by cisplatin, a cross-link agent which is widely used in treatment of lung cancer. The aim of the study is to evaluate 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: Lung squamous carcinoma SK-MES-1 cells were cultured in vitro.FANCM-si RNA and USP1-si RNA were transfected into SK-MES-1 cells using RNA interference(RNAi) technique according manufacture’s instructions. The transfection efficacy was verified by testing the FANCM and USP1 protein expression using Western blotting assay. Annexin V/PI flow cytometry was performed to measure cell apoptosis following cisplatin treatment befor and after trasfection of USP1-si RNA and FANCM-si RNA. Cell Counting Kit-8(CCK-8) technique was conducted to detect the survival of SK-MES-1 cells treated with cisplatin pre-transfection and post-transfection. Western blotting assay was used to detect the levels of FANCD2 monoubiquitination, which is indicated as ratio FANCD2-L and FANCD2-S.Immunofluorescence assay was done to determine the expression of FANCD2 nuclear foci.Result:(1) FANCM and USP1(a deubiquitinating enzyme) proteins expressions were obviously decreased after transfection of FANCM-si RNA and USP1-si RNA compared with before transfection(all P <0.05), indicating that USP1 and FANCM genes were effectively silenced in the SK-MES-1 cells.(2) Flow cytometry analysis results showed that FANCM or USP1 gene silence significantly increased ealy apoptosis of SK-MES-1 cells induced by cisplatin with concentration of 10μg/ml(P<0.05). Increase of the ealy apoptosis rate by silencing USP1 cells was more obvious than by silencing FANCM cells(P <0.05).(3) The cell survival 24 h and 48 h after treatment with cisplatin post-knockdown of FANCM or USP1 were markedly decreased compared with pre-knocdown of the two genes(P <0.05). The sensitization effect to cisplatin in cells with USP1 knockdown was more notable than in cells with FANCM knockdown(P <0.05). The survival rates of the cells after cisplatin treatment following double knocdown of FANCM and USP1 were dramaticaly lower than single knockdown of FANCM(P <0.05), and was similsr to single knocdown of USP1(P >0.05).(4) the silencing of FANCM resulted in significant down-regulation of the FANCD2 monoubiquitination and nuclear foci expression, suggesting that FANCM participate in FANCD2 monoubiquitination and foci formation(P <0.05),while the USP1 silence increased the FANCD2 monoubiquitination levels and nuclear foci expression, implying that FANCD2 deubiquitination was blocked and ubiquitinated FANCD2 were heaped up in cells(P <0.05).Conclusion: Depletion of FANCM gene by si RNA can enhance the sensitivity of lung squamous carcinoma SK-MES-1 cells to cisplatin through depression of the FA/BRCA pathway DNA repair function. Knockdown of USP1 gene by si RNA also sensitize SK-MES-1 cell to cisplatin by inhibiting deubiquitination of FANCD2 which block FA/BRCA pathway. Notably, the sensitization effect to cisplatin in silencing USP1 was more significant than in silencing FANCM.