Effects of Bax gene over-expression on expression of Smac/DIABLO in human neuroblastoma SH-SY5Y cells

HE Yun-song; ZHAO Hong-yang; XIANG Wei

doi:10.13798/j.issn.1009-153X.2014.09.009

Abstract：Objective To explore the effects of over-expression of Bcl-2-associated X protein (Bax) gene on the expression of Smac/DIABLO in human neuroblastoma (NB) cell line. Methods The human Bax gene was transfected into human NB SH-SY5Y cells with plasmid, lipofectamine 2000. The human NB SH-SY5Y cells into which empty-plasmid was transfected and the non-transfected human NB SH-SY5Y cells were served as controls. The cell clones into which objective genes had been transfected were obtained after being screened with G-418. The morphological changes of apoptotic cells were observed by Hoechst33258 fluorescent staining. Bax and Smac/DIABLO expression were determined by Western blot analysis. The intracellular distribution of Smac/DIABLO protein were evaluated by immunofluorescence (IF) assays. Results The G-418-resistant cell clones were obtained by screening continually with G-418 for 2 weeks after the transfection. Bax levels in Bax-transfected group (1.067 ± 0.036) were significantly higher than those in the empty plasmid-transfected group (0.436 ± 0.035; P<0.05) and non-transfected group (0.444 ± 0.046; P<0.05). The levels of Smac/DIABLO protein in Bax-transfected group (1.408 ± 0.044) were significantly higher than those in the empty plasmid-transfected group (0.708±0.021;P<0.05) and non-transfected group (0.748±0.041;P<0.05). But there was no significance differences in the expressions of Bax and Smac/DIABLO between both the control groups (P>0.05). IF assay showed that Smac/DIABLO was mainly localized in the cytoplasm. The typical apoptotic morphological changes were found by Hoechst33258 staining in both the Bax-transfected and non-transfected groups. The apoptotic rate of the cells in Bax-transfected group [(19.26 ± 1.11)%] was significantly higher than that in non-transfected group [(2.96 ± 0.61)%; P<0.05]. Conclusions Transfection of extrinsic Bax gene may result in its continuous over-expression in human NB SH-SY5Y cells, in which the expression of Smac/DIABLO protein may be up-regulated and the cell apotosis may be promoted by the Bax gene over-expression.