Egr-1 regulates the transcription of NGX6 gene through a Sp1/Egr-1 overlapping site in the promoter
© Liu et al.; licensee BioMed Central Ltd. 2014
Received: 19 May 2013
Accepted: 26 June 2014
Published: 16 July 2014
As a novel candidate metastasis suppressor gene, Nasopharyngeal carcinoma-associated gene 6 (NGX6) is involved in cellular growth, cell cycle progression and tumor angiogenesis. Previous studies have shown that NGX6 gene is down-regulated in colorectal cancer (CRC). However, little is known about its transcriptional regulation.
We defined the minimal promoter of NGX6 gene in a 186-bp region (from-86 to +100) through mutation construct methods and luciferase assays. Results from Electrophoretic mobility shift assays (EMSA) and Chromatin immunoprecipitation (ChIP) revealed that Early growth response gene 1 (Egr-1) binds to the Sp1/Egr-1 overlapping site of NGX6 minimal promoter. Overexpression of Egr-1 increased the promoter activity and mRNA level of NGX6 gene; while knock-down of endogenous Egr-1 decreased mRNA expression of NGX6 gene.
These results demonstrate that Egr-1 regulates NGX6 gene transcription through an overlapping Sp1/Egr-1 binding site as a positive regulator of NGX6 gene.
Nasopharyngeal carcinoma associated gene 6 (NGX6) was located on chromosome 9p21-22 . In previous studies, results from various analysis including RT-PCR, Dot hybridization and Northern blot showed that the mRNA levels of NGX6 were significantly lower in colorectal carcinoma tissues with lymph node or distant metastasis than that in paracancerous tissues . And its mRNA expression level in nasopharyngeal carcinoma tissues was also lower than that in normal nasopharyngeal epithelial tissues . Some studies demonstrated that NGX6 may play an important role in EGFR/K-ras/JNK/c-Jun/cyclinD1 signal pathway and Wnt/β-catenin signal pathway [4–6]. Overexpression of NGX6 gene in colon cancer cells was able to inhibit cell growth and cell cycle progression from G1 to S phase [7, 8]. As a transmembrane protein, NGX6 protein has been demonstrated to regulate the transduction of extracellular signals into cytoplasm and nucleus through binding with the membrane cytoskeleton-organizing protein ezrin by its cytoplasmic domain. And it is also involved in cellular adhesion, invasion, motility and metastasis [9, 10]. But its transcriptional regulation remains unknown. We have previously reported a region spanning from -159 to +276 bp as the proximal promoter of NGX6 gene . In this report, we defined the minimal promoter of NGX6 gene in a 186-bp region, and explored the role of Egr-1 in positive regulating of NGX6 expression in colon cancer cells. These results will help to further understand and uncover the bio-functions of NGX6 gene involved in the pathogenesis of colorectal carcinoma.
The human colon carcinoma cell lines, HT-29, SW480 and SW620, were from American Type Culture Collection (ATCC, Rockville, MD). COS7 cells were provided by the Cancer Research Institute, Xiangya School of Medicine, Central South University (Human, P.R. China). All cells were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and incubated at 37°C in a humidified incubator with 5% CO2.
Potential binding sites of transcription factors within the promoter region spanning from -86 to +100 bp of NGX6 gene were analyzed by MatInspector Professional (http://www.genomatix.de).
Luciferase- reporter vectors and assay
Primers used for generating NGX6 promoter constructs
Reverse: 5′- AAAGCTAGCGGGACCTGGGTAGGAGTT-3'
pGL3 + 100/+276
Forward: 5′- AAAGGTACCGCCTGAACTCCTACCCA-3'
Reverse: 5′- AAAGCTAGCGGATTGGGATAGGACGAG-3'
Forward: 5′- AAAGGTACCTGTGCTTGGGGTGAGAAA-3'
Reverse: 5′- AAAAAGCTTTTAGTCCTGCTGGGCTTC-3'
Forward: 5′- AAAGGTACCTCCTCGAAGCCCAGCAG -3'
Reverse: 5′- AAAAAGCTTACTTGACGTCGGCGTGAC-3'
pGL3 + 12/+100
Forward: 5′- AAAGGTACCGCCGACGTCAAGTCGAG-3'
Cells were seeded at 5 × 105 cells/well and cultured in 12-well plates for 24 h prior to transfection. The cells were transfected with 1 μg of various NGX6 promoter constructs, pGL3-control plasmid, or pGL3-enhancer plasmid by Lipofectamine 2000 reagent (Invitrogen) according to manufacturer’s instructions. To control transfection efficiency, cells were co-transfected with 0.5 μg SV40 β-galactosidase vector per well. 48 h after transfection, the cell lysates were prepared and luciferase activity was measured by luciferase assay kit (Promega). β-galactosidase activity was also quantified using the β-galactosidase Enzyme Assay System (Promega). Experiments were repeated at least three times with three replicates per sample.
Electrophoretic mobility shift assay (EMSA)
Nuclear protein was prepared by using NR-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Rockville, IL). Then nuclear supernatants were collected and stored at -80°C until used. Protein concentration was determined using BCA protein assay kit (Pierce Biotechnology, Rochville, IL). And the following oligonucleotides and their complementary strands were synthesized and polyacrylamide gel electrophoresis (PAGE)-purified by Songan: NGX6 (-54/-39) 5′-GTAGGGCGGGGGCG GGCTTTACT-3′ (in which the G/C-rich sequences shown in italics are potential binding sites for the transcription factors for Egr-1 and Sp1).
According to the manufacturer’s instructions of lightshift chemiluminescent EMSA Kit (Pierce), 10 ug nuclear protein extracts were incubated with 50 fmol of biotin labeled oligonucleotides for 20 min at room temperature (20°C ~ 25°C). In competition experiments, prior to the addition of the labeled oligonucleotides nuclear extracts were incubated for 10 min with excess unlabeled oligonucleotides. The reaction mixtures were then resolved on 6% polyacrylamide gel in 0.5 × TBE. Then the binding reactions were transferred to nylon membrane and crosslinking was performed with a hand-held UV lamp equipped with 254 nm bulbs. Finally, the biotin-DNA was detected by chemiluminescence.
Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed by using a kit from Upstate Biotechnology. HT-29 cells were crosslinked by ‘ing 1% formaldehyde in cell culture medium for 10 min at room temperature, followed by adding glycine to end the process. The cross-linked chromatin was sonicated to yield fragments of 200 to 1000 bp. Soluble chromatin was then clarified by centrifugation for 10 min at 14000 rpm at 4°C, and 1% of the supernatant was saved as input. Diluted soluble chromatin fragments were precleared with protein G agarose to discard nonspecifically bound chromatin fragments, then immunoprecipitated with antibodies against Sp1(07–645) (Upstate Biotechnology) or Egr-1(588) (Santa Cruz Biotechnology) overnight at 4°C. Immunocomplexes were captured on the ssDNA/protein G-agarose slurry, and washed sequentially with low-salt wash buffer, high-salt wash buffer and LiCI wash buffer, followed by two final washes with TE buffer. After washing, the immunocomplexes were eluted by incubation for 15 min at 25°C with 200 μl of elution buffer, and reversed for 6 h at 65°C. The DNA fragments were extracted with phenol/chloroform and precipitated with ethanol. The immunoprecipitated DNA samples were analyzed by PCR using the primer pair: (forward) 5′-AAAGGTACCTGTGCTTGGGGTGAGAAA-3′ and (reverse) 5′-AAAGCTAGCGGGACCTGGGTAGGAGTT-3′. PCR was carried out for 35 cycles by using a step cycle of 95°C for 30 sec, 55°C for 50 sec, 72°C for 1 min, and followed by 72°C for 10 min. A 259 bp product was detected from the reaction. As a negative control for PCR, water was added instead of the immunoprecipitated chromatin fragments. The PCR products were analyzed by electrophoresis on a 5% agarose gal.
Construction of pCMV-HA/Egr-1
To construct wild type Egr-1 expression vector, Egr-1 gene cDNA was amplified by PCR method using human fetal brain cDNA library as template. According to the sequence of Egr-1 gene (NM_001964) obtained from GeneBank, the following primers with EcoRI and XhoI restriction enzyme sites were used: Egr-1 (forward) 5′-TTTGAATTC AGGATGGCCGCGGCCAAG-3′ and (reverse) 5′-CACCTCGAG TTAGCAAATTTCAATTGTC-3′ (restriction sites underlined). After an initial denaturation step at 94°C for 10 min, the PCR was carried out for 38 cycles at 94°C for 30 sec, 58°C for 50 sec, 72°C for 2 min, and followed by 72°C for 10 min. The PCR fragments were purified and subcloned into EcoRI/XhoI-digested pCMV-HA vector, then transformed into competent JM109 cells. Positive clones were sequenced to verify the correct inserts, and then named as pCMV-HA/Egr-1.
siRNA target to Egr-1
Egr-1 oligo small interfering RNA (siRNA) (5'-CCAUGGACAACUACCCUAA[dT][dT]-3') and a negative control siRNA (5'-ACUUACGAGUGACAGUAGA[dT][dT]-3') were purchased from GenePharma. For siRNA transfection, SW480 cells were plated overnight in 6-well dish at a density of 1 × 106 cells/per well. They were transiently transfected with 100 nM siRNA using lipofectamine 2000 transfection reagent as described by the manufacturer (Invitrogen). The cell lysates were harvested 2 days after transfection. The total RNA from SW480 cells was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany). Then Egr-1 expression and NGX6 expression were evaluated by RT–PCR or Real-time quantitative PCR.
RT-PCR and Real-time quantitative PCR
Total RNA from harvested cells was isolated using Trizol reagent (Invitrogen) and then treated with DNaseI (Roche) to eliminate possible DNA contamination. RNA was quantified by optical density (A260) and stored at -80°C until used. cDNA was prepared using Reverse Transcription System (Promega).
2 μl single-stranded cDNA was amplified by PCR using NGX6-specific primers primers and glyceraldehide-3-phosphate dehydrogenase (GAPDH) primers. The GAPDH primers were added to the PCR at the end of the tenth cycle as control experiments. RT-PCR products were analyzed by electrophoresis on a 5% agarose gal.
Real-time quantitative PCR was performed using SYBR® Premix Ex Taq™ (TaKaRa, Dalian, China) according to manufacturer’s recommendations. 96 wells plates were used on BIO-RAD IQ™5 thermocycler. Cycling conditions were as follows: an initial step at 95°C 5 min for enzyme activation, followed by 40 cycles alternation of 10 sec at 95°C, 15 sec at 54°C, 30 sec at 72°C and a final dissociation step. Obtained Ct values were normalized against GAPDH. Relative gene-expression was determined by using the △Ct method . The primers were used in RT-PCR and Real-time qPCR were as follows: NGX6 forward (5'-AGAACCGCCATCCCTT-3'), NGX6 reverse (5'-CACCTCGTGAGTCAAGCA-3'), Egr-1 forward (5′-CACGAACGCCCTTACGCT-3'), Egr-1 reverse (5′-CATCGCTCCTGGCAAACT-3'), GAPDH forward (5'-AGGTCGGAGTCAACGGATTTG-3'), GAPDH reverse (5'-GTGATGGCATGGACTGTGGT-3').
Data presentation and statistics
Promoter/reporter transient transfections were repeated at least three times and results were expressed as mean ± SE. Statistical differences was determined using the Student’s t-Test. Statistical significance was determined at the p < 0.05 level. The EMSA and ChIP experiments were repeated at least three times and one representative result was shown for each set of experiments.
Identification of NGX6 minimal promoter (-86/+100)
Transcription factor Sp1 and Egr-1 bind to an overlapping Sp1/Egr-1 binding motif in the NGX6 minimal promoter
Role of the transcription factor Egr-1 in the regulation of NGX6 promoter activity
Requirement of Egr-1 for induction of NGX6 expression
NGX6 (genebank accession number AF188239), a novel candidate metastasis suppressor gene, was cloned by a location candidate cloning strategy. Its function is associated with colorectal carcinoma occurrence and development. Previous study has indicated that NGX6 was decreased or undetectable in colorectal carcinoma and involved in cellular growth, cell cycle progression and tumor angiogenesis. However, little is known about the transcriptional regulation of NGX6 gene.
Bioinformatics analysis indicated that no canonical TATA boxes were found in NGX6 promoter, while two CAAT boxes, a GpG island and putative transcription binding sites for Sp1, Egr-1, NF-Y etc. were discovered . In this report, using various promoter deletion constructs, the minimal promoter of NGX6 gene was defined in a -86 to +100 bp region, which is the shortest promoter identified so far in the regulatory region of NGX6 gene. The results of MatInspector showed that this region contained an overlapping Sp1/Egr-1 GC-rich motif for the binding of the zinc finger transcription factors Egr-1 and Sp1. EMSA along with ChIP assays confirmed a specific Sp1/Egr-1 overlapping site spanning from -54 to -39 bp in NGX6 minimal promoter. Similar Sp1/Egr-1 overlapping binding sites have been shown to play a critical role in the expression of some genes, such as TF, NDRG1 and PDGF-A[14–16]. The regulation of transcription by these two transcription factors has been shown to be complex: in some genes the two factors are synergistic, whereas in other systems the factors appear to compete [17, 18]. Egr-1 encodes a nuclear phosphoprotein that binds to the GC-rich sequence 5'-GCGGGGGCG-3' and regulates transcription of target gene through the GC-rich consensus sequence . Egr-1 expression had been found to be either decreased or undetectable in nasopharyngeal carcinoma and colorectal carcinoma [20, 21]. Various studies have indicated Egr-1 is involved in regulation of cell proliferation and may have tumor suppressive functions [22, 23]. In our experiment, immunohistochemical staining of Egr-1 showed weaker staining in metastatic tissue in comparison to non-metastatic tissue in a colorectal tissue microarray.
RT-PCR and Western Blot also confirmed that Egr-1 expression level in SW620 cells is lower than that in SW480 cells (data not shown). Therefore, we hypothesize that Egr-1 regulate the expression of NGX6 gene in colorectal cancer as a tumor suppressor gene. In our study, overexpression of Egr-1 increased the activity of NGX6 promoter and up-regulated the expression level of NGX6 mRNA, whereas knock-down of Egr-1 reduced endogenous mRNA expression of NGX6 gene in SW480 cells. From these findings, we conclude that Egr-1 is indeed a positive regulator of NGX6 gene. In previous study, a series of related experiment has revealed that nuclear transcription factor Sp1 also positively regulates NGX6 promoter transcriptional expression . Egr-1 binding may influence the occupancy of Sp1 proteins in certain environment such as hypoxia  and result in the induction of NGX6 gene expression changes. Further studies needs to be done to confirm this hypothesis.
In summary, the current study provides a molecular model for Egr-1 in positive regulation of NGX6 promoter activity and mRNA expression. These results will help to better understand the role of NGX6 gene in carcinoma progression and may provide a new potential therapeutic target for cancer therapy from the view of knockdown of Egr-1 and down-regulation of NGX6 gene.
The study was supported by grants from National Key Project of Scientific Research Program of China (2006CB910503), Hunan Province Natural Sciences Foundations of China (09JJ3066) and Postgraduate Thesis Creation Project of Central South University (2009bsxt042). National Science Foundation (81172300).
- Yang JB, Bin LH, Li ZH, Zhang XF, Qian J, Zhang BC, Zhou M, Xie Y, Deng LW, Li GY: Refined localization and cloning of a novel putative tumor suppressor gene associated with nasopharyngeal carcinoma on chromosome 9p21-22. Clin J Cancer. 2000, 19 (1): 6-9.Google Scholar
- Zhang XM, Shen SR, Wang XY, Wang JR, Li J: Expression of tumor related gene NGX6 in gastric cancer and colorectal cancer. World Chin J Digestol. 2002, 10 (8): 873-876.Google Scholar
- Ma J, Zhou J, Fan SQ, Wang LL, Li XL, Yan Q, Zhou M, Liu HY, Zhang QH, Zhou HD, Gan K, Li Z, Peng C, Xiong W, Tan C, Shen SR, Yang JB, Li J, Li GY: Role of a novel EGF-like domain-containing gene NGX6 in cell adhesion modulation in nasopharyngeal carcinoma cells. Carcinogenesis. 2005, 26 (2): 281-291.View ArticlePubMedGoogle Scholar
- Liu F, Shen SR, Li HT, Wang XY, Peng Y, Liao MT, Guo Q: Effects of NGX6 on the transcriptional activation of beta-catenin/TCF/LEF in Wnt/beta-catenin signal pathway. J Cen S Univ (Med Sci). 2007, 32 (6): 985-991.Google Scholar
- Wang XY, Shen SR, Liu F, Peng Y, Li GY, Fan SQ: Inhibitory effects of NGX6 gene on EGFR/K-ras/JNK/c-Jun/cyclin D1 signal pathway in the colon cancer. Prog Biochem Biophys. 2008, 35 (5): 570-576.Google Scholar
- Peng Y, Li HT, Wu MH, Wang XY, Fan SQ, Liu F, Xiang B, Guo Q, Tang XY, Shen SR: NGX6 inhibits AP-1 and Ets-1 expression and down-regulates cyclin D1 in human colorectal cancer. Acta Biochim Biophys Sin. 2009, 41 (6): 504-514.View ArticlePubMedGoogle Scholar
- Wang XY, Shen SR, Liu HY, Li XL, Fan SQ: Effects of NGX6 gene on cell cycle in colon cancer cell line HT-29. Prog Biochem Biophys. 2006, 33 (1): 45-50.Google Scholar
- Wang XY, Shen SR, Liu HY, Zhang XM, Peng C, Huang H, Liu F, Li XL, Li GY: Effects of tumor suppressor gene NGX6 on growth of human colon cancer cell line HT-29. World Chin J Digestol. 2004, 12 (3): 574-579.Google Scholar
- Peng SP, Li XL, Wang L, Ou-Yang J, Ma J, Wang LL, Liu HY, Zhou M, Tang YL, Li WS, Luo XM, Cao L, Tang K, Shen SR, Li GY: The role of NGX6 and its deletion mutants in the proliferation, adhesion and migration of nasopharyngeal carcinoma 5-8 F cells. Oncology. 2006, 71 (3–4): 273-281.View ArticlePubMedGoogle Scholar
- Peng S, Fan S, Li X, Wang L, Liu H, Zhou M, Wang L, Shen S, Li G: The expression of ezrin in NPC and its interaction with NGX6, a novel candidate suppressor. Cancer Sci. 2007, 98 (3): 341-349. 10.1111/j.1349-7006.2007.00410.xView ArticlePubMedGoogle Scholar
- Liu MJ, Peng Y, Wang XY, Guo Q, Shen SR, Li GY: NGX6 gene mediated by promoter methylation as a potential molecular marker in colorectal cancer. BMC Cancer. 2010, 10: 160- 10.1186/1471-2407-10-160View ArticlePubMedPubMed CentralGoogle Scholar
- Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the compareative C(T) method. Nat Protoc. 2008, 3: 1101-1108. 10.1038/nprot.2008.73View ArticlePubMedGoogle Scholar
- Liu MJ, Wang XY, Shen SR, Li N, Zhang DC, Peng Y, Guo Q, Li GY: Cloning and identification of promoter of suppressed-tumor gene NGX6. Prog Biochem Biophys. 2010, 37 (10): 1082-1089. 10.3724/SP.J.1206.2010.00102. 10.3724/SP.J.1206.2010.00102View ArticleGoogle Scholar
- Khachigian LM, Lindner V, Williams AJ, Collins T: Egr-1-induced endothelial gene expression: a common theme in vascular injury. Science. 1996, 271 (5254): 1427-1431. 10.1126/science.271.5254.1427View ArticlePubMedGoogle Scholar
- Zhang P, Tchou-Wong KM, Costa M: Egr-1 mediates hypoxia-inducible transcription of the NDRG1 gene through an overlapping Egr-1/Sp1 binding site in the promoter. Cancer Res. 2007, 67 (19): 9125-9133. 10.1158/0008-5472.CAN-07-1525View ArticlePubMedGoogle Scholar
- Midgley VC, Khachigian LM: Fibroblast growth factor-2 induction of platelet-derived growth factor-C chain transcription in vascular smooth muscle cells is ERK-dependent but not JNK-dependent and mediated by Egr-1. J Biol Chem. 2004, 279 (39): 40289-40295. 10.1074/jbc.M406063200View ArticlePubMedGoogle Scholar
- Russell S, Thomas T: Interplay between Egr-1 and Sp1critical for 13-cis retinoic acid mediated transcriptional repression of angiotensin type 1A receptor. J Mol Endocrinol. 2013, 50 (3): 361-374. 10.1530/JME-12-0154View ArticleGoogle Scholar
- Cui MZ, Penn MS, Chisolm GM: Native and oxidized low density lipoprotein induction of tissue factor gene expression in smooth muscle cells is mediated by both Egr-1 and Sp1. J Biol Chem. 1999, 274 (46): 32795-32802. 10.1074/jbc.274.46.32795View ArticlePubMedGoogle Scholar
- Liu C, Rangnekar VM, Adamson E, Merchla D: Suppression of growth and transformation and induction of apoptosis by Egr-1. Cancer Gene Ther. 1998, 5 (1): 3-18. 10.1038/sj.gt.3300598PubMedGoogle Scholar
- Zhong SJ, Yao YH, Li FH, Cai QZ: Expression and significance of EGR-1, p53, p16 and cyclinD1 in nasopharyngeal carcinoma. Cancer Res Clin. 2006, 18 (10): 664-666.Google Scholar
- Wu JW, Chen LX, Chen ZQ, Yang SH, Chen WR, Xheng ZC, Wu ZD, Zeng ZL: Theoratical significance of expression of early growth response gene-1 in rectal cancer tissues on anus-preserving operation for low rectal cancer. Chin J Clin Rehabil. 2004, 8 (23): 4786-4787.Google Scholar
- Calogero A, Lombari V, Gregorio GD, Porcellini A, Ucci A, Arcella A, Caruso R, Gagliardi FM, Gulino A, Lanzetta G, Frati L, Mercola D, Ragona G: Inhibition of cell growth by Egr-1 in human primary cultures from malignant glioma. Cancer Cell Int. 2004, 4 (1): 1- 10.1186/1475-2867-4-1View ArticlePubMedPubMed CentralGoogle Scholar
- Frank GB, Bottone J, Yuseok M, Brenda AM, Thomas E: Transcriptional regulation of activating transcription factor 3 involves the early growth response-1 gene. J Pharmacol Exp Ther. 2005, 312 (2): 668-677.Google Scholar
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