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Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells.

Guo H, Lin Y, Zhang H, Liu J, Zhang N, Li Y, Kong D, Tang Q, Ma D - BMC Mol. Biol. (2007)

Bottom Line: Tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion and metastasis.We found that both protein and mRNA of TFPI-2 could not be detected in highly invasive breast cancer cell line MDA-MB-435.In this study, we found that the CpG islands in TFPI-2 promoter was hypermethylated in highly invasive breast cancer cell line, and DNA methylation in the entire promoter region caused TFPI-2 repression by inducing inactive chromatin structure and decreasing KLF6 binding to its DNA binding sequence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Molecular Medicine, Ministry of Education, Yixueyuan Road 138#, Shanghai Medical College, Fudan University, Shanghai 200032, China. eagooselj@yahoo.com.cn

ABSTRACT

Background: Tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion and metastasis. Here, we have investigated the mechanism of DNA methylation on the repression of TFPI-2 in breast cancer cell lines.

Results: We found that both protein and mRNA of TFPI-2 could not be detected in highly invasive breast cancer cell line MDA-MB-435. To further investigate the mechanism of TFPI-2 repression in breast cancer cells, 1.5 Kb TFPI-2 promoter was cloned, and several genetic variations were detected, but the promoter luciferase activities were not affected by the point mutation in the promoter region and the phenomena was further supported by deleted mutation. Scan mutation and informatics analysis identified a potential KLF6 binding site in TFPI-2 promoter. It was revealed, by bisulfite modified sequence, that the CpG island in TFPI-2 promoter region was hypermethylated in MDA-MB-435. Finally, using EMSA and ChIP assay, we demonstrated that the CpG methylation in the binding site of KLF-6 diminished the binding of KLF6 to TFPI-2 promoter.

Conclusion: In this study, we found that the CpG islands in TFPI-2 promoter was hypermethylated in highly invasive breast cancer cell line, and DNA methylation in the entire promoter region caused TFPI-2 repression by inducing inactive chromatin structure and decreasing KLF6 binding to its DNA binding sequence.

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Expression of TFPI-2 in human breast cancer cells with different metastasis potential. (A). Western blot analysis of TFPI-2 protein expression. The HUVEC cell line was used as positive control and the protein levels of GAPDH were determined as control. (B). Quantitative real-time PCR analysis of TFPI-2 mRNA levels. All expression levels of TFPI-2 in breast cancer cell lines were normalized to the level of its expression in HUVEC. Lane 1: MDA-MB-435. Lane 2: T47D. Lane 3: MCF-7. Lane 4: HUVEC.
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Figure 1: Expression of TFPI-2 in human breast cancer cells with different metastasis potential. (A). Western blot analysis of TFPI-2 protein expression. The HUVEC cell line was used as positive control and the protein levels of GAPDH were determined as control. (B). Quantitative real-time PCR analysis of TFPI-2 mRNA levels. All expression levels of TFPI-2 in breast cancer cell lines were normalized to the level of its expression in HUVEC. Lane 1: MDA-MB-435. Lane 2: T47D. Lane 3: MCF-7. Lane 4: HUVEC.

Mentions: Expression of TFPI-2 protein in human breast cancer cell lines with different metastasis potential was examined by western blotting. As shown in Figure 1a, TFPI-2 could not be detected in highly invasive breast cancer cell line (MDA-MB-435), while it was expressed in low invasive breast cancer cell lines (MCF-7 and T47D). TFPI-2 mRNA was detected by real-time PCR and the results were corresponded with that of TFPI-2 protein expression (Figure 1b). These data indicated that the expression of TFPI-2 might be regulated at transcriptional level.


Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells.

Guo H, Lin Y, Zhang H, Liu J, Zhang N, Li Y, Kong D, Tang Q, Ma D - BMC Mol. Biol. (2007)

Expression of TFPI-2 in human breast cancer cells with different metastasis potential. (A). Western blot analysis of TFPI-2 protein expression. The HUVEC cell line was used as positive control and the protein levels of GAPDH were determined as control. (B). Quantitative real-time PCR analysis of TFPI-2 mRNA levels. All expression levels of TFPI-2 in breast cancer cell lines were normalized to the level of its expression in HUVEC. Lane 1: MDA-MB-435. Lane 2: T47D. Lane 3: MCF-7. Lane 4: HUVEC.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2233638&req=5

Figure 1: Expression of TFPI-2 in human breast cancer cells with different metastasis potential. (A). Western blot analysis of TFPI-2 protein expression. The HUVEC cell line was used as positive control and the protein levels of GAPDH were determined as control. (B). Quantitative real-time PCR analysis of TFPI-2 mRNA levels. All expression levels of TFPI-2 in breast cancer cell lines were normalized to the level of its expression in HUVEC. Lane 1: MDA-MB-435. Lane 2: T47D. Lane 3: MCF-7. Lane 4: HUVEC.
Mentions: Expression of TFPI-2 protein in human breast cancer cell lines with different metastasis potential was examined by western blotting. As shown in Figure 1a, TFPI-2 could not be detected in highly invasive breast cancer cell line (MDA-MB-435), while it was expressed in low invasive breast cancer cell lines (MCF-7 and T47D). TFPI-2 mRNA was detected by real-time PCR and the results were corresponded with that of TFPI-2 protein expression (Figure 1b). These data indicated that the expression of TFPI-2 might be regulated at transcriptional level.

Bottom Line: Tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion and metastasis.We found that both protein and mRNA of TFPI-2 could not be detected in highly invasive breast cancer cell line MDA-MB-435.In this study, we found that the CpG islands in TFPI-2 promoter was hypermethylated in highly invasive breast cancer cell line, and DNA methylation in the entire promoter region caused TFPI-2 repression by inducing inactive chromatin structure and decreasing KLF6 binding to its DNA binding sequence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Molecular Medicine, Ministry of Education, Yixueyuan Road 138#, Shanghai Medical College, Fudan University, Shanghai 200032, China. eagooselj@yahoo.com.cn

ABSTRACT

Background: Tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion and metastasis. Here, we have investigated the mechanism of DNA methylation on the repression of TFPI-2 in breast cancer cell lines.

Results: We found that both protein and mRNA of TFPI-2 could not be detected in highly invasive breast cancer cell line MDA-MB-435. To further investigate the mechanism of TFPI-2 repression in breast cancer cells, 1.5 Kb TFPI-2 promoter was cloned, and several genetic variations were detected, but the promoter luciferase activities were not affected by the point mutation in the promoter region and the phenomena was further supported by deleted mutation. Scan mutation and informatics analysis identified a potential KLF6 binding site in TFPI-2 promoter. It was revealed, by bisulfite modified sequence, that the CpG island in TFPI-2 promoter region was hypermethylated in MDA-MB-435. Finally, using EMSA and ChIP assay, we demonstrated that the CpG methylation in the binding site of KLF-6 diminished the binding of KLF6 to TFPI-2 promoter.

Conclusion: In this study, we found that the CpG islands in TFPI-2 promoter was hypermethylated in highly invasive breast cancer cell line, and DNA methylation in the entire promoter region caused TFPI-2 repression by inducing inactive chromatin structure and decreasing KLF6 binding to its DNA binding sequence.

Show MeSH
Related in: MedlinePlus