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. 2015 Sep 18;22(1):77.
doi: 10.1186/s12929-015-0185-4.

RNA interferences targeting the Fanconi anemia/BRCA pathway upstream genes reverse cisplatin resistance in drug-resistant lung cancer cells

Chun-Hua Dai  1 , Jian Li  2 , Ping Chen  3 , He-Guo Jiang  4 , Ming Wu  5 , Yong-Chang Chen  6
Affiliations

RNA interferences targeting the Fanconi anemia/BRCA pathway upstream genes reverse cisplatin resistance in drug-resistant lung cancer cells (V体育安卓版)

Chun-Hua Dai et al. J Biomed Sci. .

Abstract

Background: Cisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeutic efficacy of cisplatin is limited by the development of resistance. In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstream genes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells to cisplatin. VSports手机版.

Result: Using small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. The extent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cells was significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL can reverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted in higher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage. V体育安卓版.

Conclusion: Knockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin on suppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway V体育ios版. .

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Figures

Fig. 1
Fig. 1
The proliferation rates and FANCD2 monoubiquitination in A549 and A549/DDP cells treated with DDP. a The proliferation rates in A549/DDP cells 24 and 48 h after cisplatin treatment were significantly higher than those in A549 cells. b FANCD2 monubiquitination in A549 cells induced by cisplatin with different concentrations. c FANCD2 monubiquitination in A549/DDP cells induced by cisplatin with different concentrations. d The levels of FANCD2 monoubiquitination in A549/DDP cells were markedly higher than those in A549 cells after cisplatin treatment
Fig. 2
Fig. 2
Immunofluorescence and quantification of cells exhibiting 5 or more FANCD2 foci in lung cancer cells. Cisplatin-induced FANCD2 foci formation in A549 (a) and A549/DDP cells (b) were inhibited markedly by knockdown of FANCF, FANCL, and FANCD2. c The graph below show quantified data of FANCD2 foci in the two cells. cisplatin-induced FANCD2 foci expressions were stronger in A549/DDP than in A549 cells
Fig. 3
Fig. 3
Expression of FANCF, FANCL and FANCD2 in A549 cells after siRNA transfection. The protein and mRNA relative expression levels of FANCF (a) and FANCL (b) were the lowest by using siRNA-3 transfection among three-different siRNA transfections. c The protein and mRNA expression inhibitions of FANCD2 were the strongest by using siRNA-2 transfection among three-different siRNA transfections. BC: black control; NC: non-target control
Fig. 4
Fig. 4
The effect of FA upstream gene knockdowns on proliferation and FANCD2 monoubiquitination in A549 cells. a Knockdowns of FANCF, FANCL, or both them inhibited the proliferation rates, and (b, c) reduced FANCD2 monoubiquitination levels after cisplatin treatment. d Knockdown of FANCD2, or double knockdown of FANCD2 and FANCF, or and FANCL suppressed the proliferation rate, and (e, f) dropped FANCD2 monoubiquitination levels after cisplatin treatment
Fig. 5
Fig. 5
The effect of FA upstream gene knockdowns on proliferation and FANCD2 monoubiquitination in SK-MES-1 cells. a Knockdowns of FANCF, FANCL, or both them inhibited the proliferation rates, and (b, c) reduced FANCD2 monoubiquitination levels after cisplatin treatment. (d) Knockdown of FANCD2, or double knockdown of FANCD2 and FANCF, or and FANCL suppressed the proliferation rate, and (e, f) dropped FANCD2 monoubiquitination levels after cisplatin treatment
Fig. 6
Fig. 6
The effect of FA upstream gene knockdowns on proliferation and FANCD2 monoubiquitination in A549/DDP cells. a Knockdown of FANCF, FANCL, or both them markedly inhibited the proliferation rates, and (b, c) reduced FANCD2 monoubiquitination levels after treatment with cisplatin. (d) Knockdown of FANCD2, or double knockdown of FANCD2 and FANCF, or and FANCL significantly suppressed the proliferation rates, and (e, f) reduced FANCD2 monoubiquitination levels after treatment with cisplatin
Fig. 7
Fig. 7
Knockdown of FANCF and FANCL promote the apoptosis induced by cisplatin in A549/DDP cells. a The apoptosis rates of control cells, and (b) cells transfected with FANCF-siRNA, and (c) cells transfected with FANCL-siRNA after cisplatin treatment. Early apoptotic cells are in the lower right quadrant; late apoptotic cells are in the upper right quadrant. d The apoptosis rates in cells transfected with FANCF-siRNA or FANCL-siRNA were significantly higher than in control cells in a concentration-dependent manner. In addition, the apoptosis rates of cells transfected with FANCL-siRNA have dramatic increase as compared with cells transfected with FANCF-siRNA following treatment of 10 μg/ml and 20 μg/ml cisplatin
Fig. 8
Fig. 8
Caspase-3, caspase-9, and PARP expressions were detected by Western blot in A549/DDP cells. a The expressions of cleaved caspase-3, caspase-9, and PARP induced by cisplatin were elevated after siFANCF transfection. b The expressions of cleaved caspase-3, caspase-9, and PARP induced by cisplatin were increased after siFANCL transfection
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