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TFPI-2 is a putative tumor suppressor gene frequently inactivated by promoter hypermethylation in nasopharyngeal carcinoma.

Wang S, Xiao X, Zhou X, Huang T, Du C, Yu N, Mo Y, Lin L, Zhang J, Ma N, Murata M, Huang G, Zhang Z - BMC Cancer (2010)

Bottom Line: TFPI-2 was aberrantly methylated in 66.7% (4/6) NPC cell lines and 88.6% (62/70) of NPC primary tumors, but not in normal nasopharyngeal epithelia.TFPI-2 expression could be restored in NPC cells after demethylation treatment.Ectopic expression of TFPI-2 in NPC cells induced apoptosis and inhibited cell proliferation, colony formation and cell migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Otolaryngology Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, PR China.

ABSTRACT

Background: Epigenetic silencing of tumor suppressor genes play important roles in NPC tumorgenesis. Tissue factor pathway inhibitor-2 (TFPI-2), is a protease inhibitor. Recently, TFPI-2 was suggested to be a tumor suppressor gene involved in tumorigenesis and metastasis in some cancers. In this study, we investigated whether TFPI-2 was inactivated epigenetically in nasopharyngeal carcinoma (NPC).

Methods: Transcriptional expression levels of TFPI-2 was evaluated by RT-PCR. Methylation status were investigated by methylation specific PCR and bisulfate genomic sequencing. The role of TFPI-2 as a tumor suppressor gene in NPC was addressed by re-introducing TFPI-2 expression into the NPC cell line CNE2.

Results: TFPI-2 mRNA transcription was inactivated in NPC cell lines. TFPI-2 was aberrantly methylated in 66.7% (4/6) NPC cell lines and 88.6% (62/70) of NPC primary tumors, but not in normal nasopharyngeal epithelia. TFPI-2 expression could be restored in NPC cells after demethylation treatment. Ectopic expression of TFPI-2 in NPC cells induced apoptosis and inhibited cell proliferation, colony formation and cell migration.

Conclusions: Epigenetic inactivation of TFPI-2 by promoter hypermethylation is a frequent and tumor specific event in NPC. TFPI-2 might be considering as a putative tumor suppressor gene in NPC.

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Bisulphite genomic sequencing of NPC cells, tumor biopsies and NNEs. Bisulphite genomic sequencing of the methylation status of the 31 CpG sites within the TFPI-2 promoter in 2 NPC cell lines (CNE2 and C666-1), 2 NPC biopsies (NPC24 and NPC34) and 1NNE biopsy (NNE7). Five randomly selected clones were sequenced for each sample. Open and filled circles represent unmethylated and methylated CpG sites, respectively. Circles were partially filled according to the percentage of methylation of the CpG site. The frames show the CpG pairs covered by methylation-specific PCR primers.
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Figure 4: Bisulphite genomic sequencing of NPC cells, tumor biopsies and NNEs. Bisulphite genomic sequencing of the methylation status of the 31 CpG sites within the TFPI-2 promoter in 2 NPC cell lines (CNE2 and C666-1), 2 NPC biopsies (NPC24 and NPC34) and 1NNE biopsy (NNE7). Five randomly selected clones were sequenced for each sample. Open and filled circles represent unmethylated and methylated CpG sites, respectively. Circles were partially filled according to the percentage of methylation of the CpG site. The frames show the CpG pairs covered by methylation-specific PCR primers.

Mentions: To detect the methylation status of individual CpG sites in the NPC samples relative to the normal control biopsies, the detailed methylation status of the TFPI-2 promoter region - 178 to + 166 bp relative to the translation start site of the TFPI-2 gene was determined by bisulphite genomic sequencing in 2 NPC cell lines (CNE2 and C666-1), 2 NPC biopsies (NPC24 and NPC34) and 1 NNE sample (NNE7). The relevant 344-bp TFPI-2 promoter region contained 31 CpG sites. For each sample shown in Figure 4, the sequence of 5 representative clones is shown. All 31 CpG sites were heavily methylated in cell lines CNE2 and C666-1, in which TFPI-2 expression was silenced (Figure 4). Dense methylation of CpG dinucleotides was also observed in the 2 NPC biopsies tested. The presence of some unmethylated clones in the CNE2 and C666-1 cell lines may reflect heterogeneity in the original cell lines. The NNE samples were completely free of methylation. Bisulphite genomic sequencing also demonstrated that our bisulphite conversion of genomic DNA was complete and thus supports the reliability of our methylation-specific PCR results.


TFPI-2 is a putative tumor suppressor gene frequently inactivated by promoter hypermethylation in nasopharyngeal carcinoma.

Wang S, Xiao X, Zhou X, Huang T, Du C, Yu N, Mo Y, Lin L, Zhang J, Ma N, Murata M, Huang G, Zhang Z - BMC Cancer (2010)

Bisulphite genomic sequencing of NPC cells, tumor biopsies and NNEs. Bisulphite genomic sequencing of the methylation status of the 31 CpG sites within the TFPI-2 promoter in 2 NPC cell lines (CNE2 and C666-1), 2 NPC biopsies (NPC24 and NPC34) and 1NNE biopsy (NNE7). Five randomly selected clones were sequenced for each sample. Open and filled circles represent unmethylated and methylated CpG sites, respectively. Circles were partially filled according to the percentage of methylation of the CpG site. The frames show the CpG pairs covered by methylation-specific PCR primers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992524&req=5

Figure 4: Bisulphite genomic sequencing of NPC cells, tumor biopsies and NNEs. Bisulphite genomic sequencing of the methylation status of the 31 CpG sites within the TFPI-2 promoter in 2 NPC cell lines (CNE2 and C666-1), 2 NPC biopsies (NPC24 and NPC34) and 1NNE biopsy (NNE7). Five randomly selected clones were sequenced for each sample. Open and filled circles represent unmethylated and methylated CpG sites, respectively. Circles were partially filled according to the percentage of methylation of the CpG site. The frames show the CpG pairs covered by methylation-specific PCR primers.
Mentions: To detect the methylation status of individual CpG sites in the NPC samples relative to the normal control biopsies, the detailed methylation status of the TFPI-2 promoter region - 178 to + 166 bp relative to the translation start site of the TFPI-2 gene was determined by bisulphite genomic sequencing in 2 NPC cell lines (CNE2 and C666-1), 2 NPC biopsies (NPC24 and NPC34) and 1 NNE sample (NNE7). The relevant 344-bp TFPI-2 promoter region contained 31 CpG sites. For each sample shown in Figure 4, the sequence of 5 representative clones is shown. All 31 CpG sites were heavily methylated in cell lines CNE2 and C666-1, in which TFPI-2 expression was silenced (Figure 4). Dense methylation of CpG dinucleotides was also observed in the 2 NPC biopsies tested. The presence of some unmethylated clones in the CNE2 and C666-1 cell lines may reflect heterogeneity in the original cell lines. The NNE samples were completely free of methylation. Bisulphite genomic sequencing also demonstrated that our bisulphite conversion of genomic DNA was complete and thus supports the reliability of our methylation-specific PCR results.

Bottom Line: TFPI-2 was aberrantly methylated in 66.7% (4/6) NPC cell lines and 88.6% (62/70) of NPC primary tumors, but not in normal nasopharyngeal epithelia.TFPI-2 expression could be restored in NPC cells after demethylation treatment.Ectopic expression of TFPI-2 in NPC cells induced apoptosis and inhibited cell proliferation, colony formation and cell migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Otolaryngology Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, PR China.

ABSTRACT

Background: Epigenetic silencing of tumor suppressor genes play important roles in NPC tumorgenesis. Tissue factor pathway inhibitor-2 (TFPI-2), is a protease inhibitor. Recently, TFPI-2 was suggested to be a tumor suppressor gene involved in tumorigenesis and metastasis in some cancers. In this study, we investigated whether TFPI-2 was inactivated epigenetically in nasopharyngeal carcinoma (NPC).

Methods: Transcriptional expression levels of TFPI-2 was evaluated by RT-PCR. Methylation status were investigated by methylation specific PCR and bisulfate genomic sequencing. The role of TFPI-2 as a tumor suppressor gene in NPC was addressed by re-introducing TFPI-2 expression into the NPC cell line CNE2.

Results: TFPI-2 mRNA transcription was inactivated in NPC cell lines. TFPI-2 was aberrantly methylated in 66.7% (4/6) NPC cell lines and 88.6% (62/70) of NPC primary tumors, but not in normal nasopharyngeal epithelia. TFPI-2 expression could be restored in NPC cells after demethylation treatment. Ectopic expression of TFPI-2 in NPC cells induced apoptosis and inhibited cell proliferation, colony formation and cell migration.

Conclusions: Epigenetic inactivation of TFPI-2 by promoter hypermethylation is a frequent and tumor specific event in NPC. TFPI-2 might be considering as a putative tumor suppressor gene in NPC.

Show MeSH
Related in: MedlinePlus