Silencing of Bcl-2 by small hairpin RNA sensitizes A549 cells to radiation-induced apoptosis.

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Bcl-2 is the prominent member of a family of proteins responsiblefor dysregulation of apoptosis, and resistance to chemotherapy and radiotherapy. In this study, we investigated whether small hairpin RNA( shRNA ) targeting Bcl-2 could render A549 cells more susceptible to gamma radiation-induced apoptosis.Two of pairs oligonucleotides for shRNA expression targeting the coding region of Bcl-2 mRNA were designed and chemically synthesized. Annealed oligonucleotides were inserted into Pgenesil-1 vector with U6 promoter to construct RNAi plasmid. A negative control hairpin RNA was synthesized with a nonsense sequence. Recombinant expression vector was identified by enzyme cutting and sequencing. Bcl-2 shRNAs were transfected into A549 cells with Lipofectamine 2000. Expression of the Bcl-2 protein was assayed using immunofluorescence labeling with fluoresce isothiocyanate. The inhibition of cell growth was assessed by a MTT assay. Apoptosis was determined by Annexin-V binding assay and flow cytomertry.Identifying by enzyme cutting and sequencing showed the insertion sequence was correct. The expression levels of Bcl-2 from A549 cells decreased after Bcl-2 shRNAs treatment. There was no difference in Bcl-2 protein levels between control shRNA group and untreated cells. Viability of cells at 72 and 96 h after treatment with Bcl-2 shRNAs were less than that after treatment with control shRNAs and untreated A549 cells, respectively (P<0.05). Control shRNA had no significant effect on growth of cells. Bcl-2 shRNA combined with radiation significantly inhibited the growth of cells (P<0.05). There was no difference in cell survival treated with Bcl-2 shRNA combined with radiation at 48 h displayed changes of early apoptosis. Apoptotic rates of the A549 cells treated with Bcl-2 shRNA combined with radiation significantly increased (P<0.05), compared with either control shRNA / radiation combination or radiation-treatment cells alone. shRNAs against the Bcl-2 mRNA increases radiation-induced apoptosis in A549 cells.