ABSTRACT
Objective:To evaluate the effect of down-regulation of Sp1 expression on the radiosensitivity of glioma cells.Methods:The oligonucleotide sequence encoding shRNA was designed and synthesized, and cloned into LV3 (H1/GFP & Puro) vector to construct the recombinant. U251 and U87 cells were infected with recombinant lentivirus, then the stably-transfected cell lines were obtained by puromycin screening. The expression levels of Sp1 mRNA and protein were detected by RT-PCR and Western blot. Cell survival was detected by clonal survival assay, cell cycle was determined by flow cytometry, and DNA damage was measured by immunofluorescence assay, respectively.Results:At 72 h after infection, high expression of Sp1 lentiviral vector was observed in two cell lines under fluorescence microscope. RT-PCR and Western blot confirmed that the expression levels of Sp1 mRNA and protein were significantly down-regulated in both transfected cells (both P<0.01) and the silencing rates of Sp1 were above 90%. The sensitization enhancement ratio (SER) of shRNA-U251 and shRNA-U87 cells at 10% cell survival level were 1.39 and 1.18, respectively. After irradiation, the G 2/M phase ratio and the number of γ-H2AX foci in two Sp1 knockout groups were significantly increased. Conclusion:shRNA silencing of Sp1 increases the G 2/M phase arrest induced by X-ray, aggravates the degree of DNA double-strand breaks, and improves the radiosensitivity of glioma cells.