Silencing of ataxia-telangiectasia mutated by siRNA enhances the in vitro and in vivo radiosensitivity of glioma

Li,Yan; Li,Luchun; Li,Bo; Wu,Zhijuan; Wu,Yongzhong; Wang,Ying; Jin,Fu; Li,Dairong; Ma,Huiwen; Wang,Donglin

doi:10.3892/or.2016.4754

Silencing of ataxia-telangiectasia mutated by siRNA enhances the in vitro and in vivo radiosensitivity of glioma

Authors:
- Yan Li
- Luchun Li
- Bo Li
- Zhijuan Wu
- Yongzhong Wu
- Ying Wang
- Fu Jin
- Dairong Li
- Huiwen Ma
- Donglin Wang
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Affiliations: 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, Department of Radiotherapy, Chongqing Cancer Institute, Chongqing 400030, P.R. China
Published online on: April 19, 2016 https://doi.org/10.3892/or.2016.4754
Pages: 3303-3312

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Abstract

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.

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