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Bcl‐2 small hairpin RNAs enhance radiation‐induced apoptosis in A549 cells
Author(s) -
He DongMei,
Zhang Yuan,
Liu GeXiu
Publication year - 2007
Publication title -
cell biology international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.932
H-Index - 77
eISSN - 1095-8355
pISSN - 1065-6995
DOI - 10.1016/j.cellbi.2007.06.001
Subject(s) - small hairpin rna , transfection , lipofectamine , a549 cell , apoptosis , microbiology and biotechnology , mtt assay , cell culture , biology , western blot , chemistry , gene knockdown , recombinant dna , vector (molecular biology) , gene , biochemistry , genetics
Bcl‐2, a prominent member of the family of proteins, is responsible for dys‐regulation of apoptosis and resistance to chemotherapy and radiotherapy. This study investigated whether small hairpin RNA (shRNA) targeting Bcl‐2 could render A549 cells more susceptible to gamma radiation‐induced apoptosis. Recombinant Bcl‐2 shRNAs expression vector were transfected into A549 cells with Lipofectamine 2000. Transfected cells were screened in 800 mg/ml G418 screening medium, and after stable transfection, silencing was examined. Expression of the Bcl‐2 protein was assayed using Western blot in A549 cells. Inhibition of cell growth was assessed by a MTT assay. Apoptosis was determined by morphological observation and flow cytomertry. Expression levels of Bcl‐2 protein from A549 cells decreased after stable transfection with Bcl‐2 shRNAs. No differences in Bcl‐2 protein levels between control shRNA group and untreated cells were noted. After stable transfection with Bcl‐2 shRNAs the viability of cells was less than after stable transfection with those with control shRNAs and untransfected A549, respectively ( P < 0.05). Control shRNA had no significant effect on growth of cells. Radiation significantly inhibited the growth of cells stably transfected with Bcl‐2 shRNA ( P < 0.05). No difference in survival between the cells with control shRNA and untransfected cells was noted. Using Giemsa staining, cells stably transfected with Bcl‐2 shRNA combined with radiation at 48 h displayed changes of apoptosis. After treatment with radiation apoptotic rates of the A549 cells stably transfected with Bcl‐2 shRNA significantly increased ( P < 0.05), compared with the cells with control shRNA and untransfected cells. shRNAs against the Bcl‐2 mRNA increases radiation‐induced apoptosis in A549 cells.