Effects Of Gli1Silencing On Cisplatin Resistance Of Lung Cancer Cell A549/DDP

Objective: We transfect DDP-resistant human lung adenocarcinomacell line A549/DDP with Gli1siRNA to investigate the effects of Gli1silencing on DDP-resistance in lung cancer.Methods: At first，MTT assay was used to examine the IC50of A549and A549/DDP cells. The Gli1expression in A549and A549/DDP cells wasevaluated by qRT-PCR and Western blot. Then A549/DDP cell was culturedand transfected with Gli1siRNA. The mRNA and protein expression levelsof Gli1were evaluated by qPT-PCR, western blot analysis andimmunofluorescence microscopy. The expression of Bcl-2、caspases-3、cyclinD2was evaluated by western blot analysis. Hoechst33258stainingand flow cytometry were used to detect spontaneous cell apoptosis and cellcycle. The cell inhibition rate and DDP-induced cell death were examined byMTT and Annexin V-FITC/propidium iodide staining.Results: A549cells IC50was2.38±0.31μg/mL, while A549/DPP cellsIC50was16.75±0.54μg/mL, its resistance index was about7.04(P＜0.001). qRT-PCR and Western blot showed up-regulation of Gli1mRNA and protein expression in A549/DDP cells (P＜0.01). Gli1-knockdown by usingGli1-specific siRNA led to a markedly decrease in Gli1mRNA and proteinexpression levels, when compared to negative siRNA transfected cells anduntreated control cells (P＜0.001). The downstream effectors of Gli1, Bci-2and cyclinD2proteins were also inhibited (P＜0.01), the expression ofcaspases-3was increased (P＜0.001). Hoechst33258staining showed thatGli1depletion by Gli1siRNA in A549/DDP cells could induce spontaneousapoptosis. The result of cell cycle showed Gli1siRNA could lead more cellsarrest in G1phase. The IC50of A549/DDP cells on DDP was12.63±1.11μg/mL/13.81±1.14μg/mL, which decreased to2.65±0.85μg/mLafter being transfected with Gli1siRNA (P＜0.001). AnnexinV-FITC/propidium iodide staining showed that DDP-induced apoptosis ratein Gli1-silencing cells was higher than that in negative siRNA or untreatedcontrol cells(35.19±3.92%vs6.43±0.11%/5.01±0.77%P＜0.001).Conclusion: Application of RNA interference can restrain theexpression of GLI1mRNA and protein observably in A549/DDP cells, andincrease the sensitivity of A549/DDP cells to cisplatin. Maybe Gli1willbecome a new target to reverse the cisplatin resistance for lung cancer.