Downregulation of Survivin Gene Expression Affects Ionizing Radiation Resistance of Human T98 Glioma Cells

Abstract

Survivin is a tumor-associated gene, which has been detected in a wide variety of human tumors. Previous research has shown that Survivin can affect hepatoma carcinoma cell radiosensitivity. However, little is known about the role of Survivin in ionizing radiation resistance in glioma cells. In this study, we aimed to identify the effects of Survivin on ionizing radiation resistance in glioma cell line T98. Our results showed that downregulation of Survivin gene expression and ionizing irradiation could both inhibit T98 cell proliferation by assays in vitro including CCK-8 and immunohistochemistry. The inhibitory effect of downregulation of Survivin combined with irradiation was the most significant compared with other groups. Results of Western blotting and flow cytometric analysis also showed that downregulation of Survivin combined with the irradiation group achieved the highest apoptosis rate. Experimental results in vivo by intracranial implanting into nude mice were consistent with those in vitro. These findings indicated that ionizing radiation resistance of human T98 glioma cells can be inhibited effectively after Survivin gene silencing.

Keywords: Apoptosis; Glioma; Ionizing radiation; Proliferation; Survivin.

Fig. 1

Effects of si-RNA on the expression of Survivin gene. a The transfection efficiency was determined 3 days after incubation with lentivirus at an MOI of 2. The transfected cells labeled with GFP were observed under a fluorescence microscope (×200). b Total RNA was extracted 4 days after infection, and the relative expression of Survivin was determined using quantitative real-time PCR. GAPDH was used as an internal control. The data represent the mean ± SD of three independent experiments (**P < 0.01). c, d Total protein was extracted 4 days after infection, and the relative expression of Survivin was determined using Western blotting. GAPDH was used as an internal control. The data represent the mean ± SD of three independent experiments (**P < 0.01). GAPDH glyceraldehyde-3-phosphate dehydrogenase, GFP green fluorescence protein, MOI multiplicity of infection, PCR polymerase chain reaction, SD standard deviation

Fig. 2

Effects of downregulation of Survivin and ionizing radiation on T98 cell proliferation. a Cellular proliferation of T98 cells from different groups was measured using a CCK-8 assay daily for 3 days. b Cellular proliferation of T98 cells from different groups was measured by testing the expression of Ki-67. c The percentage of Ki-67-positive cells was calculated. Results are expressed as mean ± SD from six independent experiments (P < 0.01)

Fig. 3

Effects of downregulation of Survivin and ionizing radiation on T98 cell apoptosis. a The apoptosis rates of T98 cells from different groups were analyzed using flow cytometry. b The percentage of cells in the apoptosis phase was calculated. Results are expressed as mean ± SD from three independent experiments (P < 0.01)

Fig. 4

Downregulation of Survivin and ionizing radiation affect the expression of proteins associated with apoptosis. a, b Western blotting analysis showed that downregulation of Survivin with ionizing radiation reduced the expression of Bcl-2 most significantly while it improved the expression of Bax most significantly. Each bar represents the mean ± SD from six independent experiments (**P < 0.01)

Fig. 5

Downregulation of Survivin and ionizing radiation affect the expression of proteins associated with apoptosis in vivo. a Immunohistochemical analysis showed that downregulation of Survivin and ionizing radiation could both reduce the expression of Bcl-2 and improve the expression of Bax. However, downregulation of Survivin combined with ionizing radiation reduced the expression of Bcl-2 most significantly but improved the expression of Bax most significantly. b, c The percentages of Bax and Bcl-2 positive cells, respectively, were calculated from densitometry immunohistochemical signaling. Results are expressed as mean ± SD from three independent experiments (P < 0.01). d Imaging results showed that si-Survivin combined with ionizing radiation inhibited the tumor growth more obviously than other groups (P < 0.01)