Abstract
BackgroundMYCT1, 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.MethodsHere, 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).ResultsThe 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).ConclusionIn 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.
Highlights
MYCT1, previously named MTLC, is a novel candidate tumor suppressor gene
bisulfite-specific PCR (BSP)-based sequencing showed that 11 of the 12 CpG sites in the MYCT1 gene promoter displayed full methylation except site 12 in Hep-2 cells and laryngeal squamous cell cancer (LSCC), which indicated that the MYCT1 promoter was methylation-positive (Figure 1B)
The MYCT1 hypermethylation in LSCC was not significantly associated with age, gender, TNM staging, lymph node metastasis, distant metastasis or clinical stage of the patients (Table1)
Summary
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; the regulatory details have not been fully elucidated. Greater than 90% of laryngeal cancer has been pathologically identified as laryngeal squamous cell carcinoma (LSCC) [3]. MYCT1, which was previously named MTLC (c-Myc target from laryngeal cancer cells, GenBank access number AF527367), was cloned by our team in 2003. Fu et al in our group found that MYCT1 is transcribed at a low level in LSCC. These authors 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 binding to the E-box elements within the −886 to −655 bp region [12]
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