Irradiation enhances expression of cxcr4 in murine glioma cells via HIF-1α-independent pathway
Wei Zhou
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3
Yangyang Xu
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Xingang Li
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2
Ge Gao
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Zheng Jiang
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2
Zhenyu Shao
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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
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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,
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