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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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Related in: MedlinePlus

Effect of 5-zaz on MYCT1 expression in Hep-2 cells. A. BSP-based sequencing result of all of 12 CpG sites methlated or demethylated of in Hep-2 cells of the absence or presence of 5-aza. B. MYCT1 mRNA levels in Hep-2 cells. +5-zaz indicates Hep-2 ells were treated with 5-zaz; MYCT1 mRNA levels were significantly different.** p < 0.01.
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Figure 2: Effect of 5-zaz on MYCT1 expression in Hep-2 cells. A. BSP-based sequencing result of all of 12 CpG sites methlated or demethylated of in Hep-2 cells of the absence or presence of 5-aza. B. MYCT1 mRNA levels in Hep-2 cells. +5-zaz indicates Hep-2 ells were treated with 5-zaz; MYCT1 mRNA levels were significantly different.** p < 0.01.

Mentions: To investigate the possible epigenetic regulation of MYCT1 expression, RQ-PCR analysis was performed in Hep-2 cells that did or did not be treated with a DNA demethylating agent, 5-aza. BSP-based sequencing revealed that the MYCT1 gene promoter was demethylated in Hep-2 cells by the treatment of 5-aza (Figure 2A). Following 5-aza treatment of Hep-2 cells, MYCT1 mRNA expression increased, which showed statistically significant differences among the two groups (p < 0.01) (Figure 2B). This result suggested that DNA methylation modification down-regulated MYCT1 gene expression.


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 5-zaz on MYCT1 expression in Hep-2 cells. A. BSP-based sequencing result of all of 12 CpG sites methlated or demethylated of in Hep-2 cells of the absence or presence of 5-aza. B. MYCT1 mRNA levels in Hep-2 cells. +5-zaz indicates Hep-2 ells were treated with 5-zaz; MYCT1 mRNA levels were significantly different.** p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effect of 5-zaz on MYCT1 expression in Hep-2 cells. A. BSP-based sequencing result of all of 12 CpG sites methlated or demethylated of in Hep-2 cells of the absence or presence of 5-aza. B. MYCT1 mRNA levels in Hep-2 cells. +5-zaz indicates Hep-2 ells were treated with 5-zaz; MYCT1 mRNA levels were significantly different.** p < 0.01.
Mentions: To investigate the possible epigenetic regulation of MYCT1 expression, RQ-PCR analysis was performed in Hep-2 cells that did or did not be treated with a DNA demethylating agent, 5-aza. BSP-based sequencing revealed that the MYCT1 gene promoter was demethylated in Hep-2 cells by the treatment of 5-aza (Figure 2A). Following 5-aza treatment of Hep-2 cells, MYCT1 mRNA expression increased, which showed statistically significant differences among the two groups (p < 0.01) (Figure 2B). This result suggested that DNA methylation modification down-regulated MYCT1 gene expression.

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