摘要
目的探讨下调Sp1基因表达对胶质瘤细胞放射敏感性的影响。方法设计并合成编码shRNA的寡核苷酸序列,克隆入LV3(H1/GFP&Puro)载体中构建重组体。然后重组慢病毒分别感染U251和U87细胞,嘌呤霉素筛选得到稳定转染细胞株。荧光定量RT-PCR和蛋白质印迹法分别检测细胞中Sp1 mRNA和蛋白表达水平;克隆存活法检测细胞存活;流式法检测细胞周期;免疫荧光法检测DNA损伤程度。结果两株胶质瘤细胞在感染72 h后荧光显微镜观察Sp1慢病毒载体均高表达,RT-PCR和蛋白质印迹法结果显示转染后shRNA-U87和shRNA-U251细胞Sp1 mRMA和蛋白表达量均明显降低(P<0.01),Sp1敲除率达90%以上。shRNA-U251和shRNA-U87细胞在10%细胞存活水平下的放射增敏比(SER)分别为1.39和1.18;两株Sp1敲除组细胞经辐照后G2/M期比率及γ-H2AX foci数目与对照组相比均极显著增大。结论shRNA沉默Sp1增大了X线诱导的G2/M期阻滞,加重了DNA双链断裂程度,提高了胶质瘤细胞的放射敏感性。
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 G2/M phase ratio and the number ofγ-H2AX foci in two Sp1 knockout groups were significantly increased.Conclusion shRNA silencing of Sp1 increases the G2/M phase arrest induced by X-ray,aggravates the degree of DNA double-strand breaks,and improves the radiosensitivity of glioma cells.
作者
王琦琦
曾家豫
李强
刘雄雄
Wang Qiqi;Zeng Jiayu;Li Qiang;Liu Xiongxiong(College of Life Science,Northwest Normal University,Lanzhou 730000,China;Institute of Modern Physics,Chinese Academy of Sciences,Lanzhou 730000,China)
出处
《中华放射肿瘤学杂志》
CSCD
北大核心
2022年第7期638-642,共5页
Chinese Journal of Radiation Oncology
基金
甘肃省自然科学基金(21JR7RA097)
陇原青年创新创业人才项目(2021LQGR63)。
