Li Yan, Li Luchun, Li Bo, Wu Zhijuan, Wu Yongzhong, Wang Ying, Jin Fu, Li Dairong, Ma Huiwen, Wang Donglin
Department of Oncology, Chongqing Cancer Institute, Chongqing 400030, P.R. China.
Department of Orthopaedics, The First People's Hospital, Jiangbei, Chongqing 400020, P.R. China.
Oncol Rep. 2016 Jun;35(6):3303-12. doi: 10.3892/or.2016.4754. Epub 2016 Apr 19.
It is reported that high expression of the ataxia-telangiectasia mutated (ATM) gene is linked with radioresistance in glioma. We hypothesized that the radiosensitivity of this brain tumor is enhanced by silencing of the ATM gene. We transfected the glioma cell line U251 with the siRNA-ATMpuro (group A) lentivirus or the siRNA-HKpuro (group N, negative control) lentivirus before irradiation. RT-qPCR and western blotting were performed to verify the efficiency of siRNA‑mediated ATM silencing. Expression levels of the ATM gene and protein were obviously downregulated after transfection. Moreover, the expression of the p53, PCNA and survivin genes, which are related to radiosensitivity, was also decreased. CCK-8 and colony formation assays showed lower cell proliferation and survival in group A than in groups N and C (control group that was not transfected with any siRNA). The level of double-stranded DNA breaks was also greater in group A, as determined by the comet tail assay. Flow cytometry showed a higher rate of cell apoptosis and a higher number of cells in the G2 phase in group A. Furthermore, caspase-3, caspase-8 and caspase-9 activity was also higher in group A. In vivo analysis in mouse models created by implantation of the transfected cell lines showed that the amount of necrosis and hemorrhage was higher in group A than that in the control groups. In conclusion, silencing of ATM via the siRNA technique could improve the in vitro and in vivo radiosensitivity of glioma cells.
据报道，共济失调毛细血管扩张症突变（ATM）基因的高表达与胶质瘤的放射抗性有关。我们假设通过沉默ATM基因可增强这种脑肿瘤的放射敏感性。在照射前，我们用siRNA-ATMpuro（A组）慢病毒或siRNA-HKpuro（N组，阴性对照）慢病毒转染胶质瘤细胞系U251。进行RT-qPCR和蛋白质印迹以验证siRNA介导的ATM沉默的效率。转染后ATM基因和蛋白质的表达水平明显下调。此外，与放射敏感性相关的p53、PCNA和survivin基因的表达也降低。CCK-8和集落形成试验显示，A组的细胞增殖和存活率低于N组和C组（未转染任何siRNA的对照组）。彗星尾试验表明，A组的双链DNA断裂水平也更高。流式细胞术显示，A组的细胞凋亡率更高，G2期的细胞数量更多。此外，A组中caspase-3、caspase-8和caspase-9的活性也更高。通过植入转染细胞系建立的小鼠模型的体内分析表明，A组的坏死和出血程度高于对照组。总之，通过siRNA技术沉默ATM可提高胶质瘤细胞的体外和体内放射敏感性。
