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Promoter hypermethylation-induced transcriptional down-regulation of the gene MYCT1 in laryngeal squamous cell carcinoma.

Yang M, Li W, Liu YY, Fu S, Qiu GB, Sun KL, Fu WN - BMC Cancer (2012)

Bottom Line: 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.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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Genetics, China Medical University, Shenyang, P.R. China.

ABSTRACT

Background: 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.

Methods: 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).

Results: 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).

Conclusion: 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.

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Effect of mutation and methylation of site CGCG (−695~-692) on MYCT1 promoter activity in Hep-2 cells. A. Schematic representation of the plasmid constructs P852-mutB created by site-directed mutagenesis. The arrows indicate the primers used for constructing the mutant construct. B. Luciferase activities in Hep-2 cells of the presence or absence of 5-aza transfected with wild-type and mutant vectors of MYCT1.
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Figure 3: Effect of mutation and methylation of site CGCG (−695~-692) on MYCT1 promoter activity in Hep-2 cells. A. Schematic representation of the plasmid constructs P852-mutB created by site-directed mutagenesis. The arrows indicate the primers used for constructing the mutant construct. B. Luciferase activities in Hep-2 cells of the presence or absence of 5-aza transfected with wild-type and mutant vectors of MYCT1.

Mentions: The mutated sites of P852-mutB are illustrated in Figure 3A. The promoter region between -852 bp and -667 bp was basal for the transcriptional activity, in which the putative core promoter sequences (−730 bp to -681 bp) owned only one DNA motif of regulatory elements, a noncanonical E-box “CACGCG” [7,12]. To determine whether the CGCG site (−695 to −692) affected the promoter activity of MYCT1, we created a plasmid containing mutations in the CGCG site (−695 to −692), named P852-mutB, based on the wild-type P852 (−852 to +12). The results from a transient transfection assay showed that the P852 construct expressed a high level of luciferase activity in Hep-2 cells treated with 5-aza, indicating that some important element in the construct up-regulates MYCT1 expression. The luciferase activities in P852-mutB transfectants were significantly reduced compared with wild-type P852, which suggests that this site plays an important role in regulating MYCT1 expression (Figure 3B).


Promoter hypermethylation-induced transcriptional down-regulation of the gene MYCT1 in laryngeal squamous cell carcinoma.

Yang M, Li W, Liu YY, Fu S, Qiu GB, Sun KL, Fu WN - BMC Cancer (2012)

Effect of mutation and methylation of site CGCG (−695~-692) on MYCT1 promoter activity in Hep-2 cells. A. Schematic representation of the plasmid constructs P852-mutB created by site-directed mutagenesis. The arrows indicate the primers used for constructing the mutant construct. B. Luciferase activities in Hep-2 cells of the presence or absence of 5-aza transfected with wild-type and mutant vectors of MYCT1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472177&req=5

Figure 3: Effect of mutation and methylation of site CGCG (−695~-692) on MYCT1 promoter activity in Hep-2 cells. A. Schematic representation of the plasmid constructs P852-mutB created by site-directed mutagenesis. The arrows indicate the primers used for constructing the mutant construct. B. Luciferase activities in Hep-2 cells of the presence or absence of 5-aza transfected with wild-type and mutant vectors of MYCT1.
Mentions: The mutated sites of P852-mutB are illustrated in Figure 3A. The promoter region between -852 bp and -667 bp was basal for the transcriptional activity, in which the putative core promoter sequences (−730 bp to -681 bp) owned only one DNA motif of regulatory elements, a noncanonical E-box “CACGCG” [7,12]. To determine whether the CGCG site (−695 to −692) affected the promoter activity of MYCT1, we created a plasmid containing mutations in the CGCG site (−695 to −692), named P852-mutB, based on the wild-type P852 (−852 to +12). The results from a transient transfection assay showed that the P852 construct expressed a high level of luciferase activity in Hep-2 cells treated with 5-aza, indicating that some important element in the construct up-regulates MYCT1 expression. The luciferase activities in P852-mutB transfectants were significantly reduced compared with wild-type P852, which suggests that this site plays an important role in regulating MYCT1 expression (Figure 3B).

Bottom Line: 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.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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Genetics, China Medical University, Shenyang, P.R. China.

ABSTRACT

Background: 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.

Methods: 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).

Results: 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).

Conclusion: 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.

Show MeSH
Related in: MedlinePlus