RNA Interference-mediated FANCF Silencing Enhances The Sensitivity To Cisplatin In Lung Cancer Cells

Objective:Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenitial abnormalities, and an increased risk for cancer. Components of the FA/BRCA pathway are thought to function in the repair of DNA interstrand cross-links. Central to this pathway is the monoubiquitylation and chromatin localization of FANCD2and FANCI. To date, however, the precise mechanisms by which FA/BRCA pathway proteins process DNA lesions and decrease sensitivity of lung cancer cells to cisplatin (DDP, a DNA cross-linking agent) have not been elucidated. The aims of this study as follow:(1). To investigate relationship beween chemoresistance to cisplatin (DDP) and expression levels of FANCC, FANCF and FANCL in FA/BRCA pathway, by observing the changes in proliferation rate and the mRNA or protein levels of FANCC, FANCF and FANCL in the lung cancer cells before and after treatment with DDP. And to assess the possibility and degree of these proteins involved in the resistance to DDP, and determine the key proteins which are associated with the sensitivity to DDP in lung cancer cells.(2). To explore the effect and feasibility of increasing sensitivity to DDP in DDP-resistance A549/DDP cells by silencing FANCF or/and FANCD2genes with siRNA interference technique, and to assess correlations between FANCF gene silence and FANCD2ubiquitination levels (FANCD2-L/FANCD2-S ratio, L/S ratio) as well as the proliferation rate and apoptosis rate in lung cancer cells treated with DDP before and after FANCF transfection. Finally, to determine whether silence of FANCF or/and FANCD2genes by siRNA transfection technology can inhibit the DNA damage repair function of FA/BRCA pathway, and increase the sensitivity of lung cancer cells A549to cisplatin, and partially reverse chemoresistance to cisplatin in DDP-resistance A549/DDP cells.Methods: (1) The proliferation rates of lung cancer cells A549or SK-MES-1treated with different concentration of DDP were measured by CCK-8methods.(2) The mRNA and protein expression levels of FANCC, FANCF and FANCL were detected using RT-PCR and western blotting techniques respectively.(3) The siRNAs targeted to FANCF (FANCF-siRNAs) and FANCD2(FANCD2-siRNAs) were designed and synthesized. The siRNAs were separately or simultaneously transfected into A549and A549/DDP cells, and then the RT-PCR was used to measure the expression of FANCF and FANCD2mRNA for determining the effect of siRNA transfection.(4) The proliferation rate of the A549and A549/DDP cells treated with DDP were measureed by CCK-8methods after transfection of FANCF-siRNA and FANCD2-siRNA.(5) FANCF and FANCD2protein levels and FANCD2monoubiquitination levels, which were defined by the radio of monoubiquitination-FANCD2(L) and non-monoubiquitination-FANCD2(S), were determined using western blotting techniques in A549and A549/DDP cells treated with DDP after FANCF-siRNA or/and FANCD2-siRNA transfection.(6) The expressions of FANCD2nuclear foci in nucleus of these lung cancer cells were determined by immunofluorescence assay.(7) The apoptosis rates in these lung cancer cells were measured using flow cytometry.Results:(1) The proliferation rates were significantly decreased in A549and SK-MES-1treated with variouse concentration of DDP, with dose-depandent and time-depandent features. But the proliferation rates of SK-MES-1cells were higher than those of A549cells. Meanwhile, FANCC, FANCF and FANCL protein levels in SK-MES-1cells were significantly higher as compared with those in A549cells.(2) After tranfection of FANCF-siRNA and FANCD2-siRNA, the proliferation rates were reduced in the A549cells treated with different concentration of DDP, with dose-depandent and time-depandent features (both P<0.05). The curve changes of proliferation rate in A549cells transfected with FANCD2-siRNAalone were similar to those in A549cells transfected with FANCD2-siRNA and FANCF-siRNA simultaneously. Although the proliferation rates in the A549/DDP cells transfected with siRNA-FANCF alone were not significantly decreased after treatment of DDP, in A549/DDP cells transfected with FANCD2-siRNA and FANCF-siRNA simultaneously, the proliferation rate was markedly reduced with treatment of DDP, when compared with contral (P<0.05).(3) FANCF and FANCD2protein levels were significantly reduced in the A549cells transfected with FANCF-siRNA or/and FANCD2-siRNA after treatment of DDP (all P<0.05), with dose-depandent and time-depandent features (both P<0.05). FANCD2monoubiquitylation levels (defined as L/S ratio) in the A549cells transfected with FANCF-siRNA or/and FANCD2-siRNA were successively decrease with an increase of DDP concentration, as compared with the control (all P<0.05). In the A549/DDP cells transfected with FANCF-siRNA or/and FANCD2-siRNA, protein levels of FANCF and FANCD2were significantly reduced after treatment of different DDP compared with the control (P<0.05). Similarly, FANCD2monoubiquitylation levels (L/S ratio) were significantly reduced compared with the control (P<0.05). The bivariate correlation analysis showed that FANCF protein levels were positively correlated with FANCD2levels in A549/DDP cells with transfection of siRNA after DDP treatment (correlation coefficient r=0.90, P<0.02).(4) The apoptosis rate was significantly higher in A549/DDP cells transfected with FANCD2-siRNA and FANCF-siRNA simultaneously after treatment of different concentration DDP, with dose-dependence, when compared with the control (P<0.05). Moreover, the apoptosis rates in A549/DDP cells simultaneously transfected with FANCD2-siRNA and FANCF-siRNA were significantly higher than those in A549/DDP cells transfected with FANCD2-siRNA or FANCF-siRNA alone (P<0.05). In addition, the apoptosis rates in A549/DDP cells transfected with FANCD2-siRNA were significantly higher than those in cells transfected with FANCF-siRNA (P<0.05).(5) Immunofluorescence analysis showed that the expression of FANCD2nuclear focus was increased in A549cell and A549/DDP cells treated with DDP treatment before FANCF-siRNA transfection. After transfection with FANCD2-siRNA and FANCF-siRNA alone or simultaneously, FANCD2nuclear focus expressions in these cells were significantly reduced after DDP treatment, implying that silence of FANCF or/and FANCD2gene by siRNA transfection can reduce the DNA damage repair capacity of FA/BRCA pathway.Conclusion:(1) The resistance to DDP in SK-MES-1cells is stronger than that in A549cells, which may be associated with the higher expression levels of FANCF and FANCC. Thus, FANCF and FANCC proteins in FA/BRCA pathway are implicated in DNA damage repair and chemoresistance to DDP in lung cancer cells.(2) As a core cohesion of FA complex proteins and one of the necessary factor to maintain FA/BRCA pathway function, FANCF participate in the stability of the FA core complex and the regulation in the key step in monoubiquitylation of FANCD2after DNA damage. By silencing FANCF, the DNA damage repair function of FA/BRCA pathway is significantly inhibited. Thus FANCF is a key gene that active the FANCD2monoubiquitination, regulate the proliferation and apoptosis, and play an important rale in the mechanism of resistance to DDP in lung cancer.(3) Silencing of FANCF gene by siRNA transfection technique decreased the monoubiquitylaton levels of FANCD2, and blocked the DNA damage repair ability of FA/BRCA pathway, inhibited proliferation rate, and promoted cell apoptosis in A549and A549/DDP cells after DDP treatment. Collectively, we demonstreate that FANCF gene knockdown enhanced sensitivity to DDP in A549cells, and partially reverse resistance to DDP in A549/DDP cells. These results suggest that the inhibition of the FA/BRCA pathway is a useful adjunct to cytotoxic chemotherapy for the treatment of lung cancer.