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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 methylation of site CGCG (−695~-692) on MYCT1 promoter banding to c-Myc in Hep-2 cells. A. Binding ability of site CGCG (−695~-692) to c-Myc. End-labeled M.SssI-treated methylated and unmethylated MYCT1 promoter region probes containing CGCG (−695~-692) were mixed with nuclear proteins from Hep2 cells and separated on a 6% polyacrylamide gel. Lane 1, end-labeled M.SssI-treated methylated free probe; Lane 2, end-labeled M.SssI-treated methylated probe mixed with nuclear proteins; Lane 3,4, the unlabeled M.SssI-treated methylated wild-type competitor DNA was mixed with the nuclear proteins before adding end-labeled probe; Lane 5, c-Myc antibody was mixed with the nuclear proteins before adding end-labeled probe; B. Binding of E-box B to c-Myc in vivo detected by ChIP. The percentage of the MYCT1 promoter occupied by c-Myc was significantly different in Hep-2 cells of present or absent 5-zaz..** p < 0.01.
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Figure 4: Effect of methylation of site CGCG (−695~-692) on MYCT1 promoter banding to c-Myc in Hep-2 cells. A. Binding ability of site CGCG (−695~-692) to c-Myc. End-labeled M.SssI-treated methylated and unmethylated MYCT1 promoter region probes containing CGCG (−695~-692) were mixed with nuclear proteins from Hep2 cells and separated on a 6% polyacrylamide gel. Lane 1, end-labeled M.SssI-treated methylated free probe; Lane 2, end-labeled M.SssI-treated methylated probe mixed with nuclear proteins; Lane 3,4, the unlabeled M.SssI-treated methylated wild-type competitor DNA was mixed with the nuclear proteins before adding end-labeled probe; Lane 5, c-Myc antibody was mixed with the nuclear proteins before adding end-labeled probe; B. Binding of E-box B to c-Myc in vivo detected by ChIP. The percentage of the MYCT1 promoter occupied by c-Myc was significantly different in Hep-2 cells of present or absent 5-zaz..** p < 0.01.

Mentions: EMSA confirmed the specific binding by c-Myc antibody abrogating the band gel shift and the specific shift band was faint when the biotin-labeled probe methylated using M.SssI methylase mixed with nuclear proteins from Hep2 cells (Lane 2, 5 in Figure 4A). In the competition reaction, the specific band was not effectively competed when an excess amount of free probes methylated were added before the binding of nuclear proteins (Lane3,4 in Figure 4A). The effect of promoter methylation on the interaction between c-Myc and MYCT1 core promoter was further confirmed by ChIP assay. Using the primer to amplify c-Myc B binding regions of MYCT1 promoter, we observed different DNA levels based on the precipitate by c-Myc antibody from Hep2 cells and Hep2 cells of 5-aza treatment (Figure 4B). The quantities of DNA precipitated by c-Myc antibody from the treated Hep2 cells increased significantly (p < 0.01). These data suggest that the methylation in the core sequence including the CGCG site (−695 to −692) interferes with the specific binding of c-Myc.


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 methylation of site CGCG (−695~-692) on MYCT1 promoter banding to c-Myc in Hep-2 cells. A. Binding ability of site CGCG (−695~-692) to c-Myc. End-labeled M.SssI-treated methylated and unmethylated MYCT1 promoter region probes containing CGCG (−695~-692) were mixed with nuclear proteins from Hep2 cells and separated on a 6% polyacrylamide gel. Lane 1, end-labeled M.SssI-treated methylated free probe; Lane 2, end-labeled M.SssI-treated methylated probe mixed with nuclear proteins; Lane 3,4, the unlabeled M.SssI-treated methylated wild-type competitor DNA was mixed with the nuclear proteins before adding end-labeled probe; Lane 5, c-Myc antibody was mixed with the nuclear proteins before adding end-labeled probe; B. Binding of E-box B to c-Myc in vivo detected by ChIP. The percentage of the MYCT1 promoter occupied by c-Myc was significantly different in Hep-2 cells of present or absent 5-zaz..** p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effect of methylation of site CGCG (−695~-692) on MYCT1 promoter banding to c-Myc in Hep-2 cells. A. Binding ability of site CGCG (−695~-692) to c-Myc. End-labeled M.SssI-treated methylated and unmethylated MYCT1 promoter region probes containing CGCG (−695~-692) were mixed with nuclear proteins from Hep2 cells and separated on a 6% polyacrylamide gel. Lane 1, end-labeled M.SssI-treated methylated free probe; Lane 2, end-labeled M.SssI-treated methylated probe mixed with nuclear proteins; Lane 3,4, the unlabeled M.SssI-treated methylated wild-type competitor DNA was mixed with the nuclear proteins before adding end-labeled probe; Lane 5, c-Myc antibody was mixed with the nuclear proteins before adding end-labeled probe; B. Binding of E-box B to c-Myc in vivo detected by ChIP. The percentage of the MYCT1 promoter occupied by c-Myc was significantly different in Hep-2 cells of present or absent 5-zaz..** p < 0.01.
Mentions: EMSA confirmed the specific binding by c-Myc antibody abrogating the band gel shift and the specific shift band was faint when the biotin-labeled probe methylated using M.SssI methylase mixed with nuclear proteins from Hep2 cells (Lane 2, 5 in Figure 4A). In the competition reaction, the specific band was not effectively competed when an excess amount of free probes methylated were added before the binding of nuclear proteins (Lane3,4 in Figure 4A). The effect of promoter methylation on the interaction between c-Myc and MYCT1 core promoter was further confirmed by ChIP assay. Using the primer to amplify c-Myc B binding regions of MYCT1 promoter, we observed different DNA levels based on the precipitate by c-Myc antibody from Hep2 cells and Hep2 cells of 5-aza treatment (Figure 4B). The quantities of DNA precipitated by c-Myc antibody from the treated Hep2 cells increased significantly (p < 0.01). These data suggest that the methylation in the core sequence including the CGCG site (−695 to −692) interferes with the specific binding of c-Myc.

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