Promoter hypermethylation-induced transcriptional down-regulation of the gene MYCT1in laryngeal squamous cell carcinoma
- Min Yang†1, 2,
- Wei Li†1, 3,
- Yi-Ying Liu1,
- Shuang Fu1,
- Guang-Bin Qiu4,
- Kai-Lai Sun1 and
- Wei-Neng Fu1Email author
© Yang et al.; licensee BioMed Central Ltd. 2012
Received: 1 March 2012
Accepted: 6 June 2012
Published: 6 June 2012
MYCT1, previously named MTLC, is a novel candidate tumor suppressor gene. MYCT1 was cloned from laryngeal squamous cell cancer (LSCC) and has been found to be down-regulated in LSCC; however, the regulatory details have not been fully elucidated.
Here, we sought to investigate the methylation status of the CpG islands of MYCT1 and mRNA levels by bisulfite-specific PCR (BSP) based on sequencing restriction enzyme digestion, reverse transcription and real-time quantitative polymerase chain reaction (RQ-PCR). The function of specific sites in the proximal promoter of MYCT1 in LSCC was measured by transient transfection, luciferase assays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP).
The results suggested hypermethylation of 12 CpG sites of the promoter in both laryngeal cancer tissues and the laryngeal cancer line Hep-2 cell. The hypermethylation of the site CGCG (−695 to −692), which has been identified as the c-Myc binding site, was identified in laryngeal cancer tissues (59/73) compared to paired mucosa (13/73); in addition, statistical analysis revealed that the methylation status of this site significantly correlated with cancer cell differentiation(p < 0.01). The mRNA level of MYCT1 increased in Hep-2 cells treated with 5-aza-C (p < 0.01). The luciferase activity from mutant transfectants pGL3-MYCT1m (−852/+12, mut-695-C > A, mut-693-C > G) was significantly reduced compared with the wild type pGL3-MYCT1 (−852/+12), while the luciferase activity from wild transfectants pGL3-MYCT1 (−852/+12) rose after 5-aza treatment in Hep-2 cells. Finally, EMSA and ChIP confirmed that the methylation of the CGCG (−695 to −692) site prevented c-Myc from binding of the site and demethylation treatment of the 5′ flanking region of MYCT1 by 5-aza induced the increased occupation of the core promoter by c-Myc (p < 0.01).
In summary, this study concluded that hypermethylation contributed to the transcriptional down-regulation of MYCT1 and could inhibit cancer cell differentiation in LSCC. DNA methylation of the CGCG site (−695 to −692) of MYCT1 altered the promoter activity by interfering with its binding to c-Myc in LSCC. Epigenetic therapy of reactivating MYCT1 by 5-aza should be further evaluated in clinical trails of LSCC.
KeywordsHypermethylation MYCT1 Laryngeal squamous cell carcinoma
Laryngeal cancer is the second most common respiratory system cancer, and the overall incidence of laryngeal cancer in individuals between 0–44 years old in China has shown a highly increasing trend in the recent decades [1, 2]. Greater than 90% of laryngeal cancer has been pathologically identified as laryngeal squamous cell carcinoma (LSCC) . Currently, the primary treatment for LSCC is surgery followed by radiotherapy . Total laryngectomy, the most common type of surgery used in advanced cases, seriously impairs laryngeal function and the quality of life of LSCC patients . Therefore, understanding the epigenetic pathways involved in the pathogenesis of LSCC is still urgently needed to improve the chemotherapeutic and diagnostic molecular biomarkers to objectively appraise LSCC; in addition, a better understanding of these pathways will improve treatment and increase the survival rate of LSCC patients .
MYCT1, which was previously named MTLC (c-Myc target from laryngeal cancer cells, GenBank access number AF527367), was cloned by our team in 2003. MYCT1 is located on 6q25 and has two exons. As a putative target of c-Myc, MYCT1 is expressed in the nuclei of human hepatocellular carcinoma cells, while a wide distribution of MYCT1 has been demonstrated in various tissues . The closest homolog, mouse MT-MC1 (c-Myc target in myeloid cells-1), can regulate many c-Myc target genes in myeloid cells, which implies that like MT-MC1, MYCT1 may participate in the regulation of cell function through the c-Myc signaling network [8–10]. We previously found that MYCT1 is down-regulated in gastric cancer . Recently, Fu et al. in our group found that MYCT1 is transcribed at a low level in LSCC. These authors also confirmed that the transcriptional start site of MYCT1 is located 140 bp upstream of the ATG start codon using 5' rapid amplification of cDNA ends (RACE)(GenBank access number GU997693.1) and that c-Myc can regulate the promoter activity of MYCT1 by specifically binding to the E-box elements within the −886 to −655 bp region .
c-Myc is a transcription factor that forms heterodimers with the protein Max and binds gene promoters by recognizing the DNA sequence CACGTG, which is called a canonical E-box . c-Myc can also bind several other non-canonical E-box motifs, such as CATGTG, CATGCG, CACGCG, CACGAG, CGCGAG and CAACGTG [14–16]. Understanding the regulation of the expression of c-Myc target genes is an important step for understanding their biological functions in both physiological and pathological contexts . Several studies have demonstrated that methylation of the CpG dinucleotide within the E-box can prevent the access of c-Myc to target gene promoters in vivo[18, 19]. This idea has been further reinforced by a study that has shown that c-Myc does not bind to EGFR in HL-60 cells due to methylation of the E-box sites, whereas 5-aza-2′-deoxycytidine (5-aza), a demethylating agent that blocks cellular DNA methyltransferase activity, can restore the binding of c-Myc to the E-box and increase gene transcription . Based on these premises, a causal link exists between methylation of the E-box and c-Myc binding and transcription.
We speculate that MYCT1 might act as a tumor suppressor gene (TSG) in LSCC carcinogenesis and metastasis. The mechanism of abnormal expression of TSGs in the process of malignant transformation primarily involves point mutations, loss of heterozygosity, and epigenetic modifications (such as hypermethylation) . Specifically, aberrant methylation of CpG islands in gene-specific promoter regions can affect transcriptional function and has been found to be related to human diseases . In this study, we sought to investigate the methylation status of CpG islands, mRNA levels and the function of specific promoter sites of MYCT1 in LSCC.
Materials and methods
The human laryngeal carcinoma cell line Hep-2 was obtained from the Cell Biology Institute of Shanghai, Chinese Academy of Science. The cells were grown in RPMI-1640 with 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37 °C in a 5% CO2 humidified atmosphere.
Samples and tissue DNA extraction
Seventy-three patients who were diagnosed with resectable LSCC tumors at the ENT Department of the 463 Hospital of PLA between 2002 and 2005 were enrolled in the study. The LSCC tissues were pathologically confirmed according to the UICC classification (TNM 2002). Patients who had undergone radiotherapy or chemotherapy prior to surgery were excluded from the study. All of the patients provided written informed consent, and approval for the study was received from the Ethics Committee of China Medical University. All specimens, including cancerous and paired adjacent normal laryngeal tissues, were obtained fresh during the surgery and stored at −80 °C. Genomic DNA from the LSCC tissues and Hep-2 cells was isolated with DNAzol reagent (Invitrogen Life Technologies, San Diego, CA). Purified DNA was concentrated by ethanol precipitation. The DNA concentration was determined using a spectrophotometer.
Bisulfite modification and bisulfite-specific PCR (BSP)
Genomic DNA from Hep-2 cells and 10 LSCC samples was randomly selected for methylation status screening. Approximately 1 μg of genomic DNA was bisulfite-modified using the EZ DNA Methylation-Gold KitTM (Zymo Research, Orange, CA) according to the manufacturer's recommendations. Based on the functional promoter sequence of the MYCT1 gene, the primers (F: 5'-TTAAATAGAGAAATAGATATGTTAAGAATA-3'; R: 5'-TATACAAAATTAAAAAATAAACCAC-3') used in bisulfite-specific PCR (BSP) detection were designed using MethPrimer (http://www.urogene.org/methprimer/index.html), and the amplified fragment was 449 bp. The PCR reaction was performed in a 25 μl reaction system, starting with denaturation at 94 °C for 4 min, then 30 cycles of denaturation at 94 °C for 30 sec, annealing at 53 °C for 30 sec, extension at 72 °C for 45 sec, followed by an extra extension at 72 °C for 5 min. The BSP products (200 ng) were then cloned into a T-vector (TaKaRa, Japan), and JM109 E. coli competent cells (TaKaRa, Japan) were used for transformation according to the manufacturer’s instruction. The sequence of the BSP product was obtained using the ABI model 3730 sequencer. According to the previous study, full methylation at a particular CpG island was defined as follows: >60% of the average bisulfite sequencing signal was “C,” whereas partial methylation was 40% to 60%. “Methylation-positive” was defined as at least 6 of the 12 CpGs within the BSP product showing full or partial methylation . We then detected the methylation status of the CGCG site within the non-canonical E-box motif binding to c-Myc, CACGCG, in other LSCC tissues using BSP-based RFLP by cutting the AccII site. Briefly, 8.5 μl of BSP product digestion was initiated by adding 1 μl of 10X Buffer and 0.5 μl of 10 U/μl AccII(TaKaRa, Japan) for 6 to 7 hours in a 37 °C water bath, 5 μl of the product was analyzed on 2.5% agarose gels and the gel image was visualized under a GDS8000 (UVP, USA).
Reverse transcription and real-time quantitative polymerase chain reaction (RQ-PCR)
After culture in a 6-well plate for 24 h, Hep-2 cells were treated with 4 μM 5-aza (Sigma Chemical Co.) for 3 days as previously described . BSP was used again to detect wether Hep-2 cells had been demethylated or not. Total RNA was isolated from Hep-2 cells and Hep-2 cells treated with 5-aza using Trizol reagent (Invitrogen, USA) following the protocol. cDNA was synthesized by reverse transcription using an AMV RNA PCR kit (TaKaRa, Japan). The MYCT1 primers for RQ-PCR were F: 5'- AGGGAGTCCATGGCCAGAAA -3' and R: 5'- ATGAACACAGCCCAAATAAATCCTC -3', which amplified a 105 bp product. GAPDH served as an internal control, which primer sequences were 5'-TGCACCACCAACTGCTTAG -3' and 5'- GACGCAGGGATGATGTTC -3' and were expected to produce a 175 bp DNA fragment. RQ-PCR was carried out on a real-time quantitative PCR instrument (ABI, USA) using a TaKaRa SYBR® Premix Ex Taq™ kit (TaKaRa, Japan). The 20-μl reaction system comprised 400 nM primers, 50 ng cDNA and 10 μl SYBR Premix Ex Taq™. The cycling was designed as 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 34 s for 50 cycles. Each sample was measured in 3 separate tubes, and each experiment was run in triplicate. Results of real-time PCR were processed using a ΔΔCt method . cDNA from untreated Hep-2 cells was used as calibrator sample.
Plasmid construction and site-directed mutagenesis
We obtained a novel transcript variation of MYCT1 using 5' RACE, named c-Myc target 1 transcript variant 1(GenBank access number GU997693.1), which indicated the transcript start site of MYCT1 and its variation. Three 5' deletion constructs of p852 (−852/+12), p799 (−799/+12), and p667 (−667/+12) of the MYCT1 promoter region were generated by our group, and the results from dual luciferase assays displayed that P852 (−852/+12) acted as the proximal promoter . Based on the wild-type construct of p852, the P852 mutant was created to replace the E-box B core sequence "CACGCG" with "CAAGGG" (named P852-mutB) using the Gene Tailor site-directed mutagenesis system (Invitrogen, USA) according to the manufacturer's instructions. The primers Fp (forward primer containing site SacI, 5'-TTTGAGCTCGTGGCCGAGCGCAGT-3'), Rp (reverse primer containing site HindIII, 5'-TTTAAGCTTGTTATTAGCCATAATATCCACAAGA-3'), fm (mutated forward primer, 5'-GTGTGGTGGCAAGGGCCTGTAATCCC-3') and rm (mutated reverse primer, 5'-GGGATTACAGGCCCTTGCCACCACAC-3') were used in site-directed mutagenesis. The pMDTM-T Vector (TaKaRa, Japan) was used for TA cloning and JM109 E. coli competent cells (TaKaRa, Japan) were used for transformation. Colonies were selected and rechecked by electrophoresis. The authenticity of all constructs was confirmed by DNA sequencing on an ABI 3730xl 96-capillary DNA analyzer (ABI, US).
Transient transfection and luciferase assays
All of the cells used for the subsequent experiments were in log phase. Hep-2 cells were cultured in 24-well plates at a density of 1 × 105 per well overnight in RPMI-1640 without FBS and antibiotics. P852, P852-mutB or the empty construct of the luciferase reporter gene (0.8 μg) and 16 ng of the internal control plasmid pRL-TK were co-transfected into Hep-2 using Lipofectamine™ 2000 (Invitrogen, USA). The relative luciferase activity was normalized to the TK activity value. At 6 h after transfection, RPMI-1640 with FBS and antibiotics was added to the cells. The cells were harvested at 48 h after transfection, and the lysates were analyzed for luciferase activity using the Dual Luciferase Reporter assay (Promega) in a GloMax™ Microplate Luminometer (Promega). Hep-2 cells pre-treated with 5-aza (4 μM, 3 days) were used for P852 to be transfected. The results were expressed as relative luciferase activity. The data from triplicates out of five replicates from which the largest and the smallest values were excluded from the analysis are represented as the mean ± SD.
EMSA (electrophoretic mobility shift assay)
Two sets of complementary oligonucleotides, F: 5’-CGGGTGTGGTGGCACGCGCCTGTAATCCCA -3’ and R: 5’-TGGGATTACAGGCGCGTGCCACCACACCCG-3’ including the core sequence of CACGCG(−697 to −692), were labeled by Biotin 3' End Labeling Kit (Pierce, USA). EMSA used the Lightshift Chemiluminescent EMSA kit (Pierce, USA) according to the protocols. Nuclear extracts were prepared from Hep-2 cells using a nuclear extract kit (Active Motif, USA) as described in the instructions. The methylated probe was obtained by incubating 20 μg of the oligonucleotide with 80 units of M.SssI CpG methylase for two hours at 37 °C in vitro. Then, the mixture was heated at 65 °C for 20 min to inactivate the methylase, purified by polyacrylamide gel electrophoresis, and concentrated with Centricon 3 microconcentrators. The two types of probes (1 ng) were incubated with nuclear extracts (6 μg) in protein-DNA binding buffer (2 μl of 10X binding buffer, 1 μl of 50% Glycerol, 1 μl of MgCl2, 1 μl of poly(dI–dC), and 1 μl of 1% NP-40) on ice for 20 min. Competition reactions were pre-performed with a 40/100-fold molar excess of unlabeled, methylated double-stranded competitor DNA. For the supershift reaction, 1 μg of each anti-c-Myc antibody (N-262X, Santa Cruz) was preincubated with the nuclear extracts in the absence of poly (dI.dC) for 20 min at 4 °C. Subsequently, poly (dI.dC) was added and incubated for 5 min, followed by the addition of labeled probe for an additional 20 min. The DNA-protein complexes were separated by electrophoresis on a 6% native polyacrylamide gel for 1.5 to 2.5 h at 4 °C and 100 V, and the complexes were visualized by autoradiography.
Chromatin immunoprecipitation assay (ChIP)
ChIP assay were performed as previously described . Hep-2 cells were treated with 5-aza in accordance with RQ-PCR. Cells were fixed by the addition of 1% formaldehyde in growth medium for 10 min at 37 °C. After washed with ice-cold PBS, cells were lysed with lysis buffer containing protease inhibitors and chromatin from cells was sheared by sonicator. Half of DNA fragments was saved as input DNA. The rest was incubated with anti-c-Myc antibody (N-262X, Santa Cruz), and immunoprecipitated by protein A/G plus-agarose (Santa Cruz, USA) which was eluted and extracted with phenol-chloroform, whose concentrations were measured with a spectrophotometer. The input and immunoprecipitated DNA were used as templates for Real Time PCR performed with MYCT1 core promoter-specific primer amplifying the second c-Myc binding regions (c-Myc B, F:5'-GAGGTCAGGCCTAGTTCATG-3', and R:5'-CTTAGT CTCGCTCTGTCGC-3') in accordance with RQ-PCR except the difference of 40 cycles. The percentage of MYCT1 promoter that was bound with c-Myc B was calculated based on the differences between Ct values for input and DNA samples immunoprecipitated from Hep-2 cells treated or untreated with 5-zaz .
All statistical analyses were performed using the SPSS 13.0 software. The chi-squared test was utilized to analyze the relationship between methylation and clinicopathological characteristics. The comparisons mRAN level and percentage of MYCT1 promoter occupation were carried out using one-way analysis of variance (ANOVA) and the Students-Newman-Keuls (SNK) method were employed for group comparison. P values of less than 0.05 were considered to be statistically significant.
Methylation status of the MYCT1promoter region
Correlation of CGCG (−695~-692) methylaton with case characteristics
Paired normal tissue
Age at surgery
Lymph node metastasis
Effects of treatment with epigenetic-modifying agents on MYCT1mRNA expression
The site CGCG (−695 to −692) is involved in MYCT1promotion/enhancement
Methylation interferes with the binding between the MYCT1promoter and c-myc
Multiple genetic and epigenetic alterations in tumor suppressor genes and oncogenes are important in carcinogenesis [26–28]. Therefore, identifying the corresponding genes and exploring the regulatory mechanisms involved in tumor progression have become critical issues. DNA methylation is one of the main mechanisms of epigenetics. Methylation in gene promoter regions has been well established to contribute to the inactivation of tumor suppressors [29–31].
Our previous study revealed that low MYCT1 expression is found in LSCC and that MYCT1 can inhibit cell growth and promote cell apoptosis, which suggests that MYCT1 may be a candidate TSG . Methylation of CpG islands has been expected in the MYCT1 promoter region. The present study focused on the epigenetic alterations leading to transcriptional down-regulation of MYCT1, thereby opening a new avenue to understand the molecular pathways in laryngeal cancer and to develop potential therapeutics against laryngeal cancer.
Difference between CGCG (−695~-692) methylation and mRNA level of MYCT1 in LSCC
Methylation number (%)
MYCT1/Beta actin (ratio)
0.31 ± 0.09
P < 0.01
0.88 ± 0.15
0.25 ± 0.11
P < 0.01
0.96 ± 0.17
To confirm that the aberrant methylation of MYCT1 induced transcriptional down-regulation, we compared its mRNA levels in laryngeal carcinoma Hep2 cells to Hep2 cells treated with the inhibitor of the methylase enzyme 5-aza. We found the MYCT1 mRNA levels of MYCT1 were markedly raised (p < 0.01) after the treatment, suggesting that the gene MYCT1 was suppressed by epigenetic modification. The mechanism were found in certain tumor suppression genes of cancer cell lines [32, 33].
To explore how and why methylation of the CGCG site (−695 to −692) leads to low transcription of MYCT1, we constructed the wild-type and mutant-type expressing vectors P852 (−852/+12) and P852-mutB. The results from luciferase assays revealed that the promoter sequence including the non-canonical E-box motif CACGCG (−697 to −692) can have promoter activity and that the point mutation of cytosine at −695 and −693 impaired the activity of luciferase, suggesting that aberrations, including methylation, on this site can affect the promoter activity of MYCT1. Interestingly, with the action of 5-aza, P852 owned the higher promoter activity. In further investigation, a CGCG-containing probe was used in EMSA to identify its binding to c-Myc. The result confirmed that the non-canonical E-box motif CACGCG (−697 to −692) could bind to the nuclear extracts, the specific binding band lessened significantly because the probe was methylated, while normal labeled probes could not be competed off by the unlabeled methylated wild-type probe. In addition, we observed DNA demethylation was associated with a rising occupancy percentage of the promoter by c-Myc in ChIP assay. These data suggest that methylation around the CGCG (−695 to −692) in the MYCT1 promoter might interfere with the binding of c-Myc to this motif. According to a previous report, c-Myc controls the methylation status of the core promoter of target genes through a pathway in which DNA methyltransferase 3A is recruited depending on the cellular context . In addition, the cooperation of c-Myc and other proteins participates in the stable methylation-dependent repression of downstream genes . Consistent with our study, c-Myc could mediate transcriptional silencing through DNA methylation in cell differentiation in cancer [35, 36].
5-aza was used to select genes for early detection and prognostic biomarkers of head and neck squamous cell carcinoma (HNSCC) . Recently, 5-aza combined with other therapeutics including irradiation and chemotherapy has been considered and tried in the treatment of LSCC [38, 39].
This report is the first to describe transcriptional down-regulation of the MYCT1 gene through epigenetic pathways in malignant tumors. We conclude that MYCT1 is down-regulated by promoter hypermethylation. Our data further indicate that the transcription factor c-Myc participates in the regulation of MYCT1 transcription and that DNA methylation interferes with the binding of c-Myc to the MYCT1 promoter region. This result provides a clue for selection of LSCC patients in 5-aza-mediated treatment, but how to solve it is also a problem in further study.
This work was supported by the National Natural Science Foundation of China (81102057 and 81172577) and the Natural Science Foundation of Liaoning Province (20092110).
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