Impact Of FAAP20 And RAD51C Gene Knockdown By Sirna On The Sensitivity Of Lung Cancer Cells To Cisplatin

Objective: The FA pathway was contact in resistance of cancer cells to cisplatin by restore of DNA damage. The purpose of this experiment is to make a thorough inquiry the effect of RNA interference(RNAi) targeting FA pathway FAAP20 and RAD51 C genes on the sensitivity of human lung Calu-1 cells to cisplatin, and to study the FA pathway principle of cisplatin resistance in lung adenocarcinoma cancer cells.Methods: Small interference RNAs targeting FAAP20 and RAD51 C genes(FAAP20-si RNA and RAD51C-si RNA) were transfected into Calu-1 cells by Lipofectamine 2000 according to manufecturer’s instructions. Western blotting was used to verify si RNA trasfection efficacy, and determine the FANCD2 monoubiquitination levels induced by cisplatin, which were indicated by the radio of monoubiquitination-FANCD2(L) and non-monoubiquitination-FANCD2(S), after and before transfection. CCK-8 assay was carried out to detect the cell proliferation rate of Calu-1 cells treated with cisplatin pre-transfection and post-transfection. Flow cytometry using Annexin V/PI manner was displayed to measure cell apoptosis. Immunofluorescence stain was done to test the formation of FANCD2 nuclear foci.Results: The expressions of FAAP20 and RAD51 C proteins were dramatically decreased in Calu-1 cell treated with cisplatin after transfection of FAAP20-si RNA and RAD51 Csi RNA compared with before transfection of those(both P<0.05), demonstrating that FAAP20 and RAD51 C genes were silenced successful. The rate of cell proliferation was significantly reduced following cisplatin treatment pre- and post-transfection of the si RNAs, in a manner of concentration dependence. Knockdown of FAAP20 remarkedly down-regulated the level of FANCD2 monoubiquitination and the formation of nuclear foci in Calu-1 cells after treatment of cisplatin, indicating that FAAP20 functions in upstream of FA pathway. RAD51 C gene knockdown did not influence the level of FANCD2 monoubiquitination and nuclear foci formation, suggesting the RAD51 C work in the downstream region. The proliferation rates in Calu-1 cells were markedly declined after knockdown of either of FAAP20 and RAD51 C gene following cisplatin treatment, indicating that depletion of FAAP20 and RAD51 C increase sensitivity Calu-1 cells to cisplatin. At the same time, apoptosis rates of the cells resulted in by cisplatin were elevated after knockdown of either of FAAP20 and RAD51 C. Moreover, double knockdown of FAAP20 and RAD51 C genes further increased the cytotoxicity of cisplatin to Calu-1 cells.Conclusion: Deletion of FAAP20 and RAD51 C gene by si RNA can increase the sensitivity of human lung Calu-1 cell to cisplatin through inhibition of the FA pathway DNA damage repair. Furthermore, the sensitization effection to cisplatin was further enhanced by co-depletion of the two genes, which indicated that FAAP20 and RAD51 C gene do not completely function in a same route of the fix of DNA damage. As a result, FAAP20 and RAD51 C gene may be used as molecular aim for overcoming the resistance of lung cancer cells to cisplatin and enhancing the cure rate efficacy of lung cancer.