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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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Detection of promoter methylation of MYCT1 in LSCC and Hep-2 cells. A. Schematic representation of the location of the CpG sites and the AccII restriction site in the MYCT1 gene. Arabic numerals 1 to 12 represent the number of CpG dinucleotides in the region. B. Statistical result of DNA methylation in 10 LSCC paired samples and Hep-2 cells by BSP-based sequencing. C. Representative electrophoresis image of paired LSCC samples by BSP-based RFLP.
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Figure 1: Detection of promoter methylation of MYCT1 in LSCC and Hep-2 cells. A. Schematic representation of the location of the CpG sites and the AccII restriction site in the MYCT1 gene. Arabic numerals 1 to 12 represent the number of CpG dinucleotides in the region. B. Statistical result of DNA methylation in 10 LSCC paired samples and Hep-2 cells by BSP-based sequencing. C. Representative electrophoresis image of paired LSCC samples by BSP-based RFLP.

Mentions: Twelve CpG sites are illustrated in the sequence of the PCR product (Figure 1A). 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 LSCC, which indicated that the MYCT1 promoter was methylation-positive (Figure 1B). Both BSP-based sequencing and RFLP results (Figure 1C) at the site CGCG (−695 to −692) revealed that 46 of 73 (63%) cases of LSCC showed methylation in cancer tissues and no methylation in paired normal tissue, 13 cases showed methylation in both cancer and paired normal tissues, and 14 cases showed no methylation in either cancer or paired normal tissues. These data indicated that a significant difference in MYCT1 promoter methylation status exists between cancer and paired normal tissues (Table 1). Tumors classified as undifferentiated or poorly differentiated compared with those classified as well differentiated showed significantly higher methylation. 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).


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)

Detection of promoter methylation of MYCT1 in LSCC and Hep-2 cells. A. Schematic representation of the location of the CpG sites and the AccII restriction site in the MYCT1 gene. Arabic numerals 1 to 12 represent the number of CpG dinucleotides in the region. B. Statistical result of DNA methylation in 10 LSCC paired samples and Hep-2 cells by BSP-based sequencing. C. Representative electrophoresis image of paired LSCC samples by BSP-based RFLP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Detection of promoter methylation of MYCT1 in LSCC and Hep-2 cells. A. Schematic representation of the location of the CpG sites and the AccII restriction site in the MYCT1 gene. Arabic numerals 1 to 12 represent the number of CpG dinucleotides in the region. B. Statistical result of DNA methylation in 10 LSCC paired samples and Hep-2 cells by BSP-based sequencing. C. Representative electrophoresis image of paired LSCC samples by BSP-based RFLP.
Mentions: Twelve CpG sites are illustrated in the sequence of the PCR product (Figure 1A). 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 LSCC, which indicated that the MYCT1 promoter was methylation-positive (Figure 1B). Both BSP-based sequencing and RFLP results (Figure 1C) at the site CGCG (−695 to −692) revealed that 46 of 73 (63%) cases of LSCC showed methylation in cancer tissues and no methylation in paired normal tissue, 13 cases showed methylation in both cancer and paired normal tissues, and 14 cases showed no methylation in either cancer or paired normal tissues. These data indicated that a significant difference in MYCT1 promoter methylation status exists between cancer and paired normal tissues (Table 1). Tumors classified as undifferentiated or poorly differentiated compared with those classified as well differentiated showed significantly higher methylation. 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).

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