Irradiation enhances expression of cxcr4 in murine glioma cells via HIF-1α-independent pathway

Journal of International Medical Research, Aug 2014

Objective To investigate levels of chemokine (C–X–C motif) receptor 4 (cxcr4) mRNA and protein in X-irradiated glioma cells.

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Irradiation enhances expression of cxcr4 in murine glioma cells via HIF-1α-independent pathway

Wei Zhou 0 3 Yangyang Xu 0 1 2 Xingang Li 0 1 2 Ge Gao 0 Zheng Jiang 0 1 2 Zhenyu Shao 0 3 0 >> Version of Record - Jul 10, 2014 OnlineFirst Version of Record - Jun 4, 2014 What is This? 1 Brain Science Research Institute, Shandong University , Jinan, Shandong, China 2 Department of Neurosurgery, Qilu Hospital, Shandong University , Jinan, Shandong, China 3 Department of Radiotherapy, Cancer Centre, Qilu Hospital, Shandong University , Jinan, Shandong, China - Irradiation enhances expression of cxcr4 in murine glioma cells via HIF-1a-independent pathway Journal of International Medical Research 2014, Vol. 42(4) 926931 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0300060514533522 imr.sagepub.com Abstract Objective: To investigate levels of chemokine (CXC motif) receptor 4 (cxcr4) mRNA and protein in X-irradiated glioma cells. Methods: Murine malignant glioma GL261 cells transfected with hypoxia-inducible factor (HIF)-1a miRNA or control miRNA were irradiated with X-radiation. Cxcr4 mRNA and protein were analysed using real-time reverse transcriptionpolymerase chain reaction and Western blot, respectively. Results: Levels of cxcr4 protein in GL261 cells increased in a radiation dose-dependent manner 48 h after 0, 5, 10 and 15 Gy X-irradiation. Irradiation of both HIF-1a knockdown cells and control cells resulted in a significant increase in cxcr4 mRNA levels, compared with nonirradiated cells, at 24 h after 5 Gy X-irradiation. Conclusion: Irradiation enhances expression of cxcr4 in glioma cells via a HIF-1a-independent pathway. Introduction The main features of glioma are aggressive invasion and diffuse infiltration of tumour cells into surrounding brain tissue.1 Traditional radiotherapy involves the use of large radiation fields to ensure treatment of these diffusely invasive glioma cells, but 4Affiliated Hospital, Shandong Academy of Medical Sciences, Jinan, Shandong, China Creative Commons CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permisDsoiownnlopardoevdidfreodmtihmer.soargiegpinuabl.cwomorbkyigsuaetsttroibnuOtcetdobaesr 1s6p,e2c0if1i4ed on the SAGE and Open Access page (http://www.uk.sagepub.com/aboutus/openaccess.htm). clinical research has demonstrated that glioma tends to recur within the irradiated field.2 Radioresistance is therefore an important issue in glioma treatment. In glioma, vascular endothelial growth factor (VEGF) is upregulated by irradiation and promotes the motility of the glioma cell in vitro,3 indicating that cytokines secreted by irradiated tumour cells play a role in radioresistance. Stromal cell-derived factor 1a (SDF-1a) is the only known ligand for chemokine (CXC motif) receptor 4 (cxcr4). In glioma cells, the SDF-1a promoter is activated 24 h after exposure to 8 Gy radiation,4 and cxcr4 is a key determinant of glioma progression.5 Irradiation induces tumour satellite formation by enhancing the glioma tropism of haematopoietic progenitor cells to the tumour bulk, an effect mediated by SDF1a.4 The role of tumour cells in SDF-1a/ cxcr4-mediated radiotherapy resistance is unclear, however. Hypoxia stimulates CXCR4 production by the activation of hypoxia-inducible factor (HIF)-1a in glioma cells,6 and SDF-1a has been shown to be co-localized with HIF-1a in glioma cells that surround areas of necrosis.6 The aim of the present study was to use micro RNA (miRNA) knockdown targeting HIF-1a to investigate the role of SDF-1a/ cxcr4 signalling in radiosensitivity, in a murine malignant glioma cell line. The murine malignant glioma cell line GL261 (transfected with HIF-1a miRNA or control miRNA) was provided by the Brain Science Research Institute of Shandong University, Shandong, China. Cells were cultured at 37 C in a humidiEed 5% carbon dioxide atmosphere in Dulbeccos modified Eagles medium (DMEM; GIBCO, Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine Exponentially growing cells were irradiated at room temperature with an X-ray dose energy of 6 MV at a dose rate of 3.0 Gy/min (Varian 2300 Linear Accelerator, Palo Alto, CA, USA). Irradiation was delivered as a single dose of 5, 10 or 15 Gy on a 2-cm solid water (bolus), with a source-to-surface distance of 100 cm. Cxcr4 mRNA and protein were analysed at 24 h and 48 h after irradiation, respectively. Experiments were performed in triplicate. Real-time RTPCR The GL261 cells (5 106 cells/ml) were lysed with TRIzol reagent (Invitrogen), following the manufacturers instructions. RNA was treated with DNase and purified with the RNeasy mini kit (Qiagen, Hilden, Germany). Purified RNA was eluted in 30 ml of RNA storage solution (Ambion, Austin, TX, USA) and stored at 80 C. Total RNA was quantified by UV spectroscopy (U3000 Spectrophotometer, Hitachi, (...truncated)


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Wei Zhou, Yangyang Xu, Xingang Li, Ge Gao, Zheng Jiang, Zhenyu Shao. Irradiation enhances expression of cxcr4 in murine glioma cells via HIF-1α-independent pathway, Journal of International Medical Research, 2014, pp. 926-931, 42/4, DOI: 10.1177/0300060514533522