Radiotherapy is one of the important treatments for patients with hepatocellular carcinoma. The treatment response (or efficacy), however, is limited in many patients due to acquired radiation resistance of cancer cells. Immediate-early response 5 (IER5) is one of the genes upregulated on radiation. The gene could modulate cell cycle checkpoint, leading to a decrease of cancer cell survival in response to radiation. To better understand how IRE5 expression is regulated on radiation, this study aims to identify transcription factors that interact with IER5 promoter region in liver cancer cell line. Using bioinformatic tool, we identified promoter region of IER5 gene. Subsequent luciferase reporter assay revealed two putative GC binding factor (GCF) binding sites. We found mutations of these binding sites increased the luciferase activity, suggesting a negative regulation of GCF on IER5 transcriptional activity. The physical interaction of GCF with the gene promoter was confirmed using chromatin immunoprecipitation and electrophoretic mobility shift assay assays. Different doses of radiation were also applied in these experiments, and we found the formation of protein-DNA complex reduced with the increasing dose of radiation. Together, we propose the GCF regulated transcriptional activity, at least in part, contributed to the upregulation of IER5 on radiation. The present findings provide insights into understanding the regulatory mechanisms of IER5.
Hepatocellular carcinoma (HCC) is the most common type of malignant liver cancer. It is one of the leading causes of cancer-related deaths worldwide, and China accounts for 55% of the new HCC cases each year.1 The prognosis of HCC is poor, with 5-year survival rate <10%, mainly due to high incidences of tumor recurrence and metastasis after surgery.2 Currently, non-surgical treatments for HCC include: trans-hepatic artery chemoembolization, absolute alcohol injection, radiofrequency ablation, radiotherapy and multi-target tyrosine kinase inhibitors. Among these treatments, radiotherapy is becoming important, given the recent advances in 3D conformal techniques that permit accurate delivery of high doses of radiation to the cancerous tissue. Acquired resistance of cancer cells, however, still limits the therapeutic efficiency of radiotherapy.
Efforts have been made to identify genes that are responsible for governing radiosensitivity of HCC cells. Our team and other research groups employed microarray analysis and discovered that immediate-early response 5 (IER5) was one of the genes that upregulated after treatment with radiation.3, 4, 5 Kis et al.4 found that IER5 was upregulated in human primary fibroblasts, and the level of upregulation was depended on the dose and duration of radiation. Further investigation of IER5 expression using quantitative real-time PCR in HeLa cells and in human lymphoblastoid AHH-1 cells also revealed the rapid induction of IER5 messenger RNA after exposure to radiation.6
IER5 belongs to slow-kinetics IER genes. The induction of these genes is much slower after stimulation, when compared with the fast-kinetic subclass of the immediate-early genes. IER5 could be induced by stimulation with growth factors like FGF or PDGF.7 It has also been reported that IER5 was upregulated during waking and sleep deprivation in cerebral cortex of rats,8 and was upregulated in the brains of mouse embryos exposed to teratogenic valpronic acid that could cause the failure of neural tube closure in newborn mice.9 Peptide bound polysaccharides could induce apoptosis in human promyelotic leukemic HL-60 cells, and in these cells IER5 was found upregulated,10 suggesting the role of IER5 in apoptosis. A suppression of IER5 by RNA interference resulted in an increase of cell proliferation and induced G2/M arrest.6 A study from our team showed that silencing of IER5 enhanced subcutaneous tumor growth and rendered tumor resistance to radiation (unpublished data). Together, these results indicated that IER5 plays an important role in radiation-mediated cell death and cell cycle checkpoints. Despite these findings, the detailed mechanism of radiation-induced IRE5 expression remains to be fully elucidated.
In this study, we aim to understand the transcriptional regulation of IER5 by characterizing the promoter region of IER5 and identifying transcription factors that interact with the promoter region. These findings would provide insights into understanding the development of radiation resistance of cancer cells.
Materials and methods
Cell culture and radiation exposure
Human HCC cell line HepG2 was cultured in Dulbecco’s minimal essential medium (Gibco/Brl, USA) supplemented with 10% fetal bovine serum. For each experiment, cells were exposed to cobalt-60 γ-ray at doses of 0, 2 or 4 Gy.
Reporter constructs containing wild type or mutant promoter region of IER5 gene
The DNA sequence of human IER5 gene (accession number: NM_016545) was retrieved from the GenBank of National Center for Biotechnology Information. Nucleotide sequence covering 8742 bp upstream to IER5 gene was identified as the promoter region; while the potential transcription factors were predicted using PROSCAN (http://www-bimas.cit.nih.gov/molbio/proscan).
To generate the constructs for luciferase reporter assay, fragment flanking −408 to −238 bp of the IER5 was amplified using PCR and subsequently cloned into pGL3-basic vector (the construct was called pGL3-IER5-luc/171 hereafter). Point mutations at the putative transcription factor-binding sites on the promoter region were inserted using QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) following the manufacturer’s instruction. The wild-type construct pGL3-IER5-luc/171 served as the template. The synthetic oligonucleotides harboring the desired mutations are as listed in Table 1. All constructs containing point mutations were confirmed by DNA sequencing.
Luciferase reporter assay
HepG2 cells cultured in 24-well plates were transfected with wild type or mutant reporter constructs and pRL-TK vector (as an internal control) in serum-free medium using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). After 48 h, the promoter activity was assessed using Dual-Luciferase reporter assay system (Promega, Madison, WI, USA). All experiments were performed in triplicate with positive (pGL3-Control, Promega) and negative (pGL3-Basic, Promega) controls. Student’s t-test was used to compare differences between groups.
Electrophoretic mobility shift assay
Nuclear protein extracts were prepared, and the protein concentration was measured. Double-stranded oligonucleotide probes were synthesized and labeled with biotin. Nuclear extract (17 μg) was incubated with unlabeled oligonucleotide probes or CGF polyclonal antibody in 20 μl 1x binding buffer for 10 min at room temperature, followed by addition of labeled probes and 20 min of incubation at room temperature. The reaction products were then resolved on a 4.5% non-denaturing polyacrylamide gel. The DNA was transferred to a positive nylon membrane and UV cross-linked (120 J for 40 s). LightShift electrophoretic mobility shift assay Detection kit (ThermoFisher, Pierce, USA) was then used to detect the signals.
Chromatin immunoprecipitation assay
HepG2 cells exposed to radiation were cross-linked using 1% formaldehyde at 37 °C for 10 min and resuspended in 200 μl sodium dodecyl sulfate lysis buffer. The cells were then sonicated, followed by centrifugation. Portion of the supernatant was collected as a positive input control. The rest of the lysate was diluted in buffer containing protease inhibitor. Half of the diluted supernatant was collected as a negative control before addition of antibodies. GCF antibody was added to another half of the supernatant and incubated overnight at 4 °C, and then recovered with salmon sperm DNA/protein A agarose. Precipitates were washed and incubated for 15 min with 250 μl of elution buffer (1% sodium dodecyl sulfate, 0.1 M sodium bicarbonate). The elute was reverse cross-linked by heating at 65 °C for 4 h with 20 μl of 5 M NaCl. The DNA was recovered and amplified by PCR using primers targeting GCF gene.
The DNA was recovered and amplified by PCR with forward primer 1 5′-IndexTermGACTAACGTTCGAACGGGCC-3′ or forward primer 2 5′-IndexTermCGTGATTGGCGGTCGAGAAG-3′ and reverse primer 5′-IndexTermGCGCGCTGCTCTACTAACCT- 3′.
Identification of four transcription factor-binding sites on IER5 gene promoter
IER5 gene promoter region was amplified by PCR and cloned into pGL3-basic reporter vector.
Bioinformatics analysis identified four transcription factor-binding sites on the promoter region of IER5 gene: two separate sites for GCF, and another two for nuclear factor I and transthyretin-inverted-repeat, respectively (Figure 1). The function of these transcription factors in modulating IER5 expression was confirmed through point mutation analysis. Mismatch was introduced into respective binding sites to prevent binding of the transcription factors to the promoter. Totally five reporter constructs were generated: pGL3-IER5-luc/171, wild-type IER5 promoter; mutant 1, mutated GCF-binding site; mutant 2, mutated nuclear factor I-binding site; mutant 3, mutated transthyretin inverted-repeat-binding site; and mutant 4, mutated site of another GCF.
Functional characterization of transcription factor-binding sites
The constructs were transfected into HepG2, followed by exposure to 60Co gamma-rays in the dose of 0, 2 or 4 Gy. Luciferase reporter assays showed that HepG2, co-transfected with mutant 1 or mutant 4, had a significant higher level of luminescence signals when compared with the control (transfected with pGL3-IER5-luc/171) exposed to the same dose of gamma-rays (P<0.05), suggesting a negative regulation of GCF on IER5 promoter activity. Mutant 2, on the other hand, gave a significantly lower signal than control (P<0.05), suggesting a positive regulation of nuclear factor I on IER5 promoter activity. No significant differences were found in reporter activity between mutant 3 and control, indicating transthyretin-inverted-repeat may not contribute to the regulation of IER5 transcriptional activity. In addition, increased luminescence signals were observed with increasing dose of gamma-rays in HepG2 transfected with mutant 1 or mutant 4, implicating the suppression of GCF on IER5 promoter activity reduced with the increasing dose of gamma-rays. On the other hand, the positive regulation of nuclear factor I appeared increased with the increasing dose of gamma-rays (Figure 2).
Binding of GCF to IER5 promoter in vitro
We used electrophoretic mobility shift assay to investigate whether GCF is physically bound to the IER5 promoter in vitro. Two GCF probes were synthesized to cover the putative binding sites from −397 to −372 and from −283 to −258. The bands representing the probe-protein complex were detected (Figure 3a, lanes 3, 6, 9 and 12). A reduction in intensity or elimination of the bands were observed on the addition of 200-fold unlabeled probes (Figure 3a, lanes 2, 5, 8 and 11). The addition of anti-GCF also reduced the intensity of the bands (Figure 3a, lanes 4, 7, 10 and 13), suggesting the binding between GCF protein and IER5 promoter sequence at −397 to −372 was specific. Similar results were observed using GCF probes covering IER5 promoter sequence at −283 to −258, indicating these two region of IER5 promoter form complex with GCF protein (Figure 3b).
Furthermore, among the cells exposed to increasing levels of radiation (0, 2 and 4 Gy), the intensity of the bands of both putative binding sites decreased, suggesting radiation could interfere the protein-DNA binding, which may explain why the expression of IER5 increased on radiation.
Binding of GCF to IER5 promoter in vivo
Chromatin immunoprecipitation assay was performed to investigate the DNA-protein complex in vivo. After immunoprecipitation by anti-GCF antibody, the DNA fragments associated with the GCF protein were recovered and amplified by PCR. The positive signals observed on the gel electrophoresis suggest the GCF-IER5 promoter interaction (Figures 4 and 5, lanes 4, 7 and 10). In addition, the intensity of the bands decreased with the increasing level of radiation (0, 2 and 4 Gy), indicating the protein-DNA binding was weaken on radiation, which may lead to the release of the suppressive effect of GCF on IER5 expression.
Expression of IER5 is upregulated in cells exposed to radiation,4, 6, 9, 10 and the upregulation leads to G2/M cell cycle arrest and suppression of cell proliferation.6, 11 Despite the findings, the mechanism that modulates IER5 expression in response to radiation, especially in cancer cells, remains to be fully elucidated. In this context, we characterized the promoter of IER5 gene in HepG2, demonstrating for the first time that GCF interacted with the promoter region of IER5, and such interaction could repress the transcriptional activity of IER5. Notably, we also revealed that the protein-DNA complex formation was reduced with the increasing dose of radiation. The reduction in complex formation could be due to the reduction of GCF expression on radiation, or due to the translocation of GCF in cell (such as from nucleus to cytoplasm). Further investigation will be needed to elucidate the mechanism behind. Together, we proposed that the repressor GCF could inhibit transcriptional activity of IER5 through binding to the two regions (−397 to −372 and −283 to −258) at the IER5 promoter. Within a certain range of radiation doses, the inhibitory effect of GCF on IER5 transcriptional activity was reduced as the dose of radiation increased. IER5, thereby, is upregulated in cell on radiation treatment. A recent study reported that transcriptional activity of IER5 could be regulated by heat shock factor-1 in HeLa cells.12 Further investigation will be needed to understand whether heat shock factor-1 also regulate IER5 gene in HCC.
GCF was found highly expressed in cell lines derived from T-cell lymphoma, followed by gastric carcinomas, melanoma and myeloma.13 It has been shown that GCF could bind to the GC-rich sequences of the promoter region of epidermal growth factor receptor, and is able to repress its transcriptional activity.14 epidermal growth factor receptor serves an important role in normal cell growth and development. Overexpression of the receptor was often observed in malignant than in normal tissue.15 The potential mechanistic links between radiation, IER5, and epidermal growth factor receptor via GCF warrants further investigations.
Sensitivity of HCC cells to radiation is a key to the efficacy of radiotherapy. A cascade of molecular events, such as apoptosis and cell cycle point, are involved in regulating cell survival after exposure to radiation. The current study elucidated the upregulation of one of the key genes, IER5, on radiation, and such finding will help understanding the regulatory mechanisms that contribute to radiosensitivity of HCC cells.
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This study was supported by The National Natural Science Foundation of China (Grant No’s. 30371232; and 30770533) and The National Basic Research Program of MOST, China (973 Program, Grant No. 2007CB914603).
The authors declare no conflict of interest.