Impacts of Gli1 silencing on cisplatin resistance of lung cancer cell A549/DDP
10.3969/j.issn.1000-484X.2014.08.013
- VernacularTitle:沉默Gli1基因对人非小细胞肺癌A549／DDP顺铂耐药性的影响
- Author:
Lijun DA
;
Hua LI
;
Xianquan ZHANG
- Publication Type:Journal Article
- Keywords:
Gli1 gene;
Lung cancer;
RNAi;
Cisplatin resistance;
Apoptosis
- From:
Chinese Journal of Immunology
2014;(8):1064-1068
- CountryChina
- Language:Chinese
-
Abstract:
To investigate the role of Gli1 in DDP-resistance in lung cancer.Methods:A DDP-resistant human lung adenocarcinoma cell line A 549/DDP was cultured and transfected with Gli 1 siRNA.The mRNA and protein expression levels of Gli 1 were evaluated by qPT-PCR,Western blot analysis and immunofluorescence microscopy.The expression of Bcl-2、caspases-3、cyclinD1 was evaluated by Western blot analysis.Hoechst 33258 staining and flow cytometry were used to detect spontaneous cell apoptosis and cell cycle.The cell inhibition rate and DDP-induced cell death were examined by MTT and Annexin V-FITC/propidium iodide staining.Results:Gli1-knockdown by using Gli 1-specific siRNA led to a markedly decrease in Gli 1 mRNA and protein expression levels,when compared to negative siRNA transfected cells and untreated control cells (P=0.001).The downstream effectors of Gli1, Bcl-2 and cyclinD1 proteins were also inhibited (P=0.003),the expression of caspase-3 was increased (P=0.001).Hoechst 33258 staining showed that Gli1 depletion by Gli1 siRNA in A549/DDP cells could induce spontaneous apoptosis.The result of cell cycle showed Gli1 siRNA could lead more cells arrest in G1 phase.The IC50 of A549/DDP cells on DDP was (12.63 ±1.11 )μg/ml /(13.81±1.14)μg/ml,which decreased to (2.65±0.85)μg/ml after being transfected with Gli1 siRNA (P=0.000).Annexin V-FITC/propidium iodide staining showed that DDP-induced apoptosis rate in Gli 1-silencing cells was higher than that in negative siRNA or untreated control cells ( 35.19%±3.92% vs 6.43%±0.11%/5.01%±0.77%, P=0.000 ).Conclusion: Application of RNA interference can restrain the expression of Gli 1 mRNA and protein observably in A549/DDP cells,and increase the sensitivity of A549/DDP cells to cisplatin.Maybe Gli1 will become a new target to reverse the cisplatin resistance for lung cancer.
