BACKGROUND
human tissue factor pathway inhibitor- <dig>  is a kunitz-type serine proteinase inhibitor synthesized and secreted into extrocelluar matrix  by endothelial cells, smooth muscle cells, fibroblasts, keratinocytes and urothelium  <cit> .
tfpi- <dig> readily inhibits trypsin, plasmin, chymotrypsin, cathepsin g, plasma kallikrein and the factor viia-tissue factor complex, but not urokinasetype plasminogen activator , tissue-type plasminogen activator  or thrombin  <cit> .
it had already been reported that the expression of tfpi- <dig> was down regulated in several invasive tumor cell lines, including choriocarcinoma, glioma, prostate cancer, melanoma and fibrosarcoma, moreover ectopic expression of this gene inhibits tumors growth and metastasis in vivo by regulating pericellular ecm remodeling and angiogenesis  <cit> .
nevertheless, the mechanisms that alter/modify the expression of tfpi- <dig> gene in cancer cells are not well understood.
cytosine hypermethylation at cpg dinucleotides in the promoter of tumor suppressor genes represents a major mechanism for gene inactivation in cancer.
methylation at 5' position of cytosine has been reported to alter or interfere with the correct binding of transcription factors to target sequences overlapping cpg dinucleotides  <cit> , and it also has a positive effect to recruit methyl-cpg binding activities that associate with histone deacetylases and other chromtin-modifying elements that lead to a transcriptionally silenced state.
many genes are hypermethylated at their cpg islands-containing promoters and subsequently inactivate in human tumors of different etiology  <cit> .
tfpi- <dig> promoter exhibits typical features of a housekeeping gene with a high gc-rich content .
it has a typical gc box known as binding site for the transcription factor sp <dig>  and three transcription initiation sites  binding site by bioinformatics.
as transcription factors, klf <dig> and sp <dig> cooperatively transactivate the endoglin promoter of collagen alpha <dig>  upa, tgf-beta <dig>  and tgf-beta receptor type  <dig>  <cit> .
direct physical interaction between sp <dig> and klf <dig> was documented by coimmunoprecipitation, pull-down experiments, and the gal <dig> one-hybrid system, mapping the klf <dig> interaction to the c-terminal domain of sp <dig>  <cit> .
breast cancer is the most common malignancy among females.
hypermethylation of promoter cpg islands, which is frequently observed in breast cancer  <cit> , is often associated with transcriptional silencing of the associated gene.
in this paper, we explored both genetic and epigenetic mechanisms controlling tfpi- <dig> expression in human breast cancer cells and the results indicated that tfpi- <dig> expression could be silenced by promoter hypermethylation by inducing inactive chromatin structure and decreasing klf <dig> binding to its dna binding sequence.
RESULTS
expression of tfpi- <dig> in breast cancer cells
expression of tfpi- <dig> protein in human breast cancer cell lines with different metastasis potential was examined by western blotting.
as shown in figure 1a, tfpi- <dig> could not be detected in highly invasive breast cancer cell line , while it was expressed in low invasive breast cancer cell lines .
tfpi- <dig> mrna was detected by real-time pcr and the results were corresponded with that of tfpi- <dig> protein expression .
these data indicated that the expression of tfpi- <dig> might be regulated at transcriptional level.
the critical region for tfpi- <dig> promoter activity in breast cancer cell lines
to determine if genetic variations contribute to the down regulation of tfpi- <dig> in breast cancer cells, the potential promoter region  of tfpi- <dig> was amplified from various cell lines including normal human umbilical vein endothelial cell , low and high metastatic breast cancer cell lines.
after sequencing, several genetic variations were detected among breast cancer cell lines , while the sequence from huvec was consistent with what kamei s had reported   <cit> .
except + <dig> a to g in mda-mb- <dig>  all genetic variations lie in the distal region of tfpi- <dig> promoter.
the constructs of tfpi- <dig> promoter luciferase-reporter bearing respective genetic variations were transiently transfected into breast cancer cells and the luciferase activities were measured according to the manufacture's instruction.
as shown in the figure 2a, the luciferase activities of these constructs showed only slight difference and were not consistent with tfpi- <dig> expression pattern in these cell lines, indicating that the genetic variations located in the region from - <dig> to - <dig> had no apparent effects on its promoter activities.
to find out the critical region of tfpi- <dig> promoter in breast cancer cell lines, we constructed several deletion constructs  and transiently transfected them into mcf- <dig> cell line.
the construct containing - <dig> to - <dig> showed stronger luciferase activity, suggesting the activator/enhancer were presence between - <dig> to - <dig>  the transactivation effects were suppressed by the construct containing nucleotides - <dig> to - <dig>  indicating that the repressor/silencer located in this region.
in addition, the constructs - <dig> to - <dig> exhibited virtually the same luciferase activity as the - <dig> construct, suggesting that nucleotides between - <dig> to + <dig> contain the sufficient promoter sequence for the expression of human tfpi- <dig> gene in breast cancer cell .
to further explore the functional relevance of tfpi- <dig> promoter sequences within - <dig> to + <dig>  we generated linker-scan mutants in this region.
as shown in figure 2c, the luciferase activities of sm <dig> to sm <dig> were obviously decreased , indicating - <dig> to - <dig> is critical for tfpi- <dig> promoter activity.
analysis of this essential region by matinspector revealed four most possible transcription binding sites including ap <dig>  ap <dig>  klf <dig> and sp <dig>  <cit> .
in addition, the mutation sm <dig> and sm <dig> decreased the promoter activity most significantly  and this region contains the core matrix of klf <dig>  indicating that klf <dig> plays an important role in the regulation of tfpi- <dig> in breast cancer cells.
klf  <dig> can bind and transactivate the promoter of tfpi-2
we cotransfected p- <dig> promoter-luciferase constructs with various amounts of klf <dig> expression plasmids.
as shown in figure 3a, the transcription activity of p- <dig> increased 3~ <dig> folder than control accordance with the increasing of klf <dig>  to further test whether klf <dig> binds to - <dig> ~ - <dig> region, we performed electrophoretic mobility shift assays  using the predicated klf <dig> binding site as probe with nuclear extracts prepared from breast cancer cells.
as shown in fig 3b, the probe formed one specific band, consistent with an authentic klf <dig> binding motif  and antibodies to klf <dig> produced super shifted complexes.
there was no difference of klf <dig> binding capability between mda-mb- <dig> and mcf- <dig> cell lines .
effects of dna hypermethylation on the expression of tfpi-2
the  <dig> bp dna fragment ranging from - <dig>  <dig> to + <dig>  <dig> of the human tfpi- <dig> gene was analyzed for cpg islands using online program  <cit> .
after setting the criteria of length > <dig> nt, with an observed to expected cpg dinucleotide ratio > <dig>  and g+c continent >50%, two cpg-rich islands were found .
the methylation status of the first cpg islands, encompassing - <dig> to + <dig>  was determined by bisulfite sequencing.
as shown in figure 4a, all cpg dinucleotides in the region from - <dig> to + <dig>  except - <dig> and - <dig> sites, was methylated in mda-mb- <dig> cells, while in mcf- <dig> cells, no methylation of cpg was.
together with the scan mutation data, we concluded that the binding of klf <dig> was affected by cpg methylation, resulting in silence of tfpi- <dig> in mda-mb- <dig> cell line.
treatment of mda-mb- <dig> with 5-aza-2'-deoxycitidine, the most commonly used demethylation agent, obviously restored tfpi- <dig> expression in a concentration-dependent manner , indicating that the down regulation of tfpi- <dig> in breast cancer cells is related with dna hypermethylation.
cpg hypermethylation diminished the binding of klf <dig> to tfpi- <dig> promoter
to test the effects of cpg methylation on the binding of klf <dig>  we carried out the competition experiment.
in detail, the formation of klf <dig> and kc complex was competed with different concentration  of cold kc, k <dig>  mk <dig> and kmute.
as shown in figure 5a, formation of klf <dig> complex was abrogated more effectively by competition with k6-50× and kc-50× than that by competition with mk6-50× and kmute-50×, indicating the transcription activator klf <dig> has higher affinity with unmethylated probe than cpg methylated oligonucleotides, and the nucleotides c in the core matrix of klf <dig> binding site was important to klf <dig> binding for mutation of c to t decreased the binding affinity obviously.
in addition, we used in vivo chip assays to demonstrate the effects of cpg methylation on klf <dig> binding.
klf <dig> could bind to tfpi- <dig> promoter within the cpg islands in mcf- <dig> cells, for which the tfpi- <dig> promoter is unmethylated, but not in mda-mb- <dig> cells, for which this promoter is methylated .
these data suggested that klf <dig> could bind to cpg islands in tfpi- <dig> promoter and diminished by cpg methylation in its core sequence.
discussion
our study showed that human breast cancer cells exhibited a differential pattern of tfpi- <dig> expression, with high invasive breast cancer cells  showing no expression of tfpi- <dig> at all, while much higher levels of tfpi- <dig> expression were observed in lower invasive mcf- <dig> and t47d.
all three bands reacted with anti-tfpi- <dig> polyclone antibody were single-gene products with differential glycosylation  <cit> .
it had already been reported that tfpi- <dig> had an anti-invasive effect that might be mediated via inhibition of plasmin that activates proteases promoting degradation of extracellular matrix and tumor invasion.
several tumor cell lines were less invasive when they were stably transfected with tfpi- <dig>  <cit> .
these results suggest that repression of tfpi- <dig> gene may be an important event that contributes to breast cancer progression as well as its malignant and invasive phenotype.
despite the clonal origin of most tumors, their tremendous heterogeneity suggests that cancer progression springs from the combined forces of both genetic and epigenetic events, which produce variant clonal populations, together with the selective pressures of the microenvironment, which promote growth and, perhaps, dissemination of variants with a specific set of characteristics  <cit> .
we hypothesized that both genetic variations and dna hypermethylation might contribute to the down regulation of tfpi- <dig> during the progression of breast cancer.
to verify this hypothesis, we focused on two cell lines, mcf- <dig>  a hormone-sensitive and low invasive cell line that represents early stage human breast cancer, and mda-mb- <dig> cell, which are hormone-insensitive and represent late stage breast cancer  <cit> .
it had already been reported that no sequence variations were detected in any coding exon of tfpi- <dig> gene in blood donors and apoplectic patients, while the promoter region is relatively polymorphic  <cit> .
we cloned the potential promoter region  of tfpi- <dig> gene and found several genetic mutations among breast cancer cell lines.
however, these variations located in the region from - <dig> to - <dig> had no apparent effects on their luciferase reporter activities.
one possible explanation as why the genetic alteration at a specific locus does not have an impact on its transcriptional activity was the distal region of tfpi- <dig> promoter where the point mutation located was not critical for tfpi- <dig> promoter activity  <cit> .
the deletion and scan mutation of tfpi- <dig> promoter support this idea.
cancers often exhibit aberrant methylations at gene promoter regions that is associated with loss of gene function  <cit> .
tfpi- <dig> promoter exhibits typical features of a housekeeping gene  <cit> .
analysis with methprimer revealed that two complete cpg islands spanning promoter and exon  <dig> and  <dig>  <cit> .
cpg island hypermethylation has been described in almost every tumor type including breast cancer  <cit> .
it had also been reported that dnmt inhibitor restored expression of tfpi- <dig> in several cancer cell lines  <cit> .
methylation at the 5' position of cytosine has been reported to alter or interfere with the correct binding of transcription factors to target sequences overlapping cpg dinucleotides  <cit> , and it also recruit methyl-cpg binding activities that associate with histone deacetylases and other chromtin-modifying elements that lead to a transcriptionally silenced state.
klf <dig>  a candidate tumor suppressor gene in prostate cancer  <cit> , contains  <dig> aa and is present in many tissues, including placenta, heart, lung, liver and pancreas.
as an activator of transcription, klf <dig> interacts with the core promoter element of a tata box-less gene  <cit>  and cooperate with other transcription factors in kruppel-like family, including sp <dig> and klf <dig>  <cit> .
it had already been well documented that the promoter region of tfpi- <dig> has a high gc-rich content , with the presence of typical gc boxes known as binding sites for the transcription factor sp <dig>  <cit> .
after we found that there were no difference of klf <dig> binding between mda-mb- <dig> and mcf- <dig> cell , we considered that the cooperation of klf <dig> and sp <dig> might be affected by abnormal hypermethylation in klf <dig> binding site.
treating the cell line mda-mb- <dig> with trichostatin a  which is a histone deacetylase inhibitor alone  could not reactivate expression of tfpi- <dig>  but tsa could enhance the effects of methylase inhibitor aza .
the tsa insensitive component of dnmt <dig> repression may be mediated by its interaction with dnmt associated protein   <cit> .
these data indicated a cooperative model for histone deacetylation and dna methylation in maintaining the silence of tfpi- <dig> in high invasive breast cancer cells.
another question needed to be addressed is why the sp <dig> binding site in tfpi- <dig> promoter remains unmethylated in high metastasis breast cancer cell.
a possible proposal is that sp <dig> elements protect a cpg island from de novo methylation, occurred in tumor genesis  <cit>  and this needs to be further investigated.
CONCLUSIONS
in the present work, we shown that: 1) expression of tfpi- <dig> was repressed in highly invasive breast cancer cell lines; 2) the - <dig> ~ - <dig> region was critical for tfpi- <dig> promoter activity and a klf <dig> binding site existed in this region which was abnormally hypermethylated in mda-mb-435; 3) the cpg methylation in the binding site of klf- <dig> blocked the binding of klf <dig> to tfpi- <dig> promoter, diminished the trans-activation of klf <dig> on the expression of tfpi- <dig> gene.
on the basis of these findings, we conclude that the down regulation of tfpi- <dig> in highly metastasis breast cancer was due to the abnormal hypermethylation in cpg dinucleotides of klf- <dig> binding site.

 human tissue factor pathway inhibitor- is a kunitz-type serine proteinase inhibitor synthesized and secreted into extrocelluar matrix by endothelial cells, smooth muscle cells, fibroblasts, keratinocytes and urothelium. tfpi- readily inhibits trypsin, plasmin, chymotrypsin, cathepsin g, plasma kallikrein and the factor viia-tissue factor complex, but not urokinasetype plasminogen activator, tissue-type plasminogen activator or thrombin. it had already been reported that the expression of tfpi- was down regulated in several invasive tumor cell lines, including choriocarcinoma, glioma, prostate cancer, melanoma and fibrosarcoma, moreover ectopic expression of this gene inhibits tumors growth and metastasis in vivo by regulating pericellular ecm remodeling and angiogenesis. nevertheless, the mechanisms that alter/modify the expression of tfpi- gene in cancer cells are not well understood. cytosine hypermethylation at cpg dinucleotides in the promoter of tumor suppressor genes represents a major mechanism for gene inactivation in cancer. methylation at 5' position of cytosine has been reported to alter or interfere with the correct binding of transcription factors to target sequences overlapping cpg dinucleotides, and it also has a positive effect to recruit methyl-cpg binding activities that associate with histone deacetylases and other chromtin-modifying elements that lead to a transcriptionally silenced state. many genes are hypermethylated at their cpg islands-containing promoters and subsequently inactivate in human tumors of different etiology. tfpi- promoter exhibits typical features of a housekeeping gene with a high gc-rich content. it has a typical gc box known as binding site for the transcription factor sp and three transcription initiation sites binding site by bioinformatics. as transcription factors, klf and sp cooperatively transactivate the endoglin promoter of collagen alpha upa, tgf-beta and tgf-beta receptor type. direct physical interaction between sp and klf was documented by coimmunoprecipitation, pull-down experiments, and the gal one-hybrid system, mapping the klf interaction to the c-terminal domain of sp. breast cancer is the most common malignancy among females. hypermethylation of promoter cpg islands, which is frequently observed in breast cancer, is often associated with transcriptional silencing of the associated gene. in this paper, we explored both genetic and epigenetic mechanisms controlling tfpi- expression in human breast cancer cells and the results indicated that tfpi- expression could be silenced by promoter hypermethylation by inducing inactive chromatin structure and decreasing klf binding to its dna binding sequence.  expression of tfpi- in breast cancer cells expression of tfpi- protein in human breast cancer cell lines with different metastasis potential was examined by western blotting. as shown in figure 1a, tfpi- could not be detected in highly invasive breast cancer cell line, while it was expressed in low invasive breast cancer cell lines. tfpi- mrna was detected by real-time pcr and the results were corresponded with that of tfpi- protein expression. these data indicated that the expression of tfpi- might be regulated at transcriptional level. the critical region for tfpi- promoter activity in breast cancer cell lines to determine if genetic variations contribute to the down regulation of tfpi- in breast cancer cells, the potential promoter region of tfpi- was amplified from various cell lines including normal human umbilical vein endothelial cell, low and high metastatic breast cancer cell lines. after sequencing, several genetic variations were detected among breast cancer cell lines, while the sequence from huvec was consistent with what kamei s had reported. except + a to g in mda-mb- all genetic variations lie in the distal region of tfpi- promoter. the constructs of tfpi- promoter luciferase-reporter bearing respective genetic variations were transiently transfected into breast cancer cells and the luciferase activities were measured according to the manufacture's instruction. as shown in the figure 2a, the luciferase activities of these constructs showed only slight difference and were not consistent with tfpi- expression pattern in these cell lines, indicating that the genetic variations located in the region from - to - had no apparent effects on its promoter activities. to find out the critical region of tfpi- promoter in breast cancer cell lines, we constructed several deletion constructs and transiently transfected them into mcf- cell line. the construct containing - to - showed stronger luciferase activity, suggesting the activator/enhancer were presence between - to - the transactivation effects were suppressed by the construct containing nucleotides - to - indicating that the repressor/silencer located in this region. in addition, the constructs - to - exhibited virtually the same luciferase activity as the - construct, suggesting that nucleotides between - to + contain the sufficient promoter sequence for the expression of human tfpi- gene in breast cancer cell. to further explore the functional relevance of tfpi- promoter sequences within - to + we generated linker-scan mutants in this region. as shown in figure 2c, the luciferase activities of sm to sm were obviously decreased, indicating - to - is critical for tfpi- promoter activity. analysis of this essential region by matinspector revealed four most possible transcription binding sites including ap ap klf and sp. in addition, the mutation sm and sm decreased the promoter activity most significantly and this region contains the core matrix of klf indicating that klf plays an important role in the regulation of tfpi- in breast cancer cells. klf can bind and transactivate the promoter of tfpi-2 we cotransfected p- promoter-luciferase constructs with various amounts of klf expression plasmids. as shown in figure 3a, the transcription activity of p- increased 3~ folder than control accordance with the increasing of klf to further test whether klf binds to - ~ - region, we performed electrophoretic mobility shift assays using the predicated klf binding site as probe with nuclear extracts prepared from breast cancer cells. as shown in fig 3b, the probe formed one specific band, consistent with an authentic klf binding motif and antibodies to klf produced super shifted complexes. there was no difference of klf binding capability between mda-mb- and mcf- cell lines. effects of dna hypermethylation on the expression of tfpi-2 the bp dna fragment ranging from - to + of the human tfpi- gene was analyzed for cpg islands using online program. after setting the criteria of length > nt, with an observed to expected cpg dinucleotide ratio > and g+c continent >50%, two cpg-rich islands were found. the methylation status of the first cpg islands, encompassing - to + was determined by bisulfite sequencing. as shown in figure 4a, all cpg dinucleotides in the region from - to + except - and - sites, was methylated in mda-mb- cells, while in mcf- cells, no methylation of cpg was. together with the scan mutation data, we concluded that the binding of klf was affected by cpg methylation, resulting in silence of tfpi- in mda-mb- cell line. treatment of mda-mb- with 5-aza-2'-deoxycitidine, the most commonly used demethylation agent, obviously restored tfpi- expression in a concentration-dependent manner, indicating that the down regulation of tfpi- in breast cancer cells is related with dna hypermethylation. cpg hypermethylation diminished the binding of klf to tfpi- promoter to test the effects of cpg methylation on the binding of klf we carried out the competition experiment. in detail, the formation of klf and kc complex was competed with different concentration of cold kc, k mk and kmute. as shown in figure 5a, formation of klf complex was abrogated more effectively by competition with k6-50× and kc-50× than that by competition with mk6-50× and kmute-50×, indicating the transcription activator klf has higher affinity with unmethylated probe than cpg methylated oligonucleotides, and the nucleotides c in the core matrix of klf binding site was important to klf binding for mutation of c to t decreased the binding affinity obviously. in addition, we used in vivo chip assays to demonstrate the effects of cpg methylation on klf binding. klf could bind to tfpi- promoter within the cpg islands in mcf- cells, for which the tfpi- promoter is unmethylated, but not in mda-mb- cells, for which this promoter is methylated. these data suggested that klf could bind to cpg islands in tfpi- promoter and diminished by cpg methylation in its core sequence. discussion our study showed that human breast cancer cells exhibited a differential pattern of tfpi- expression, with high invasive breast cancer cells showing no expression of tfpi- at all, while much higher levels of tfpi- expression were observed in lower invasive mcf- and t47d. all three bands reacted with anti-tfpi- polyclone antibody were single-gene products with differential glycosylation. it had already been reported that tfpi- had an anti-invasive effect that might be mediated via inhibition of plasmin that activates proteases promoting degradation of extracellular matrix and tumor invasion. several tumor cell lines were less invasive when they were stably transfected with tfpi-. these results suggest that repression of tfpi- gene may be an important event that contributes to breast cancer progression as well as its malignant and invasive phenotype. despite the clonal origin of most tumors, their tremendous heterogeneity suggests that cancer progression springs from the combined forces of both genetic and epigenetic events, which produce variant clonal populations, together with the selective pressures of the microenvironment, which promote growth and, perhaps, dissemination of variants with a specific set of characteristics. we hypothesized that both genetic variations and dna hypermethylation might contribute to the down regulation of tfpi- during the progression of breast cancer. to verify this hypothesis, we focused on two cell lines, mcf- a hormone-sensitive and low invasive cell line that represents early stage human breast cancer, and mda-mb- cell, which are hormone-insensitive and represent late stage breast cancer. it had already been reported that no sequence variations were detected in any coding exon of tfpi- gene in blood donors and apoplectic patients, while the promoter region is relatively polymorphic. we cloned the potential promoter region of tfpi- gene and found several genetic mutations among breast cancer cell lines. however, these variations located in the region from - to - had no apparent effects on their luciferase reporter activities. one possible explanation as why the genetic alteration at a specific locus does not have an impact on its transcriptional activity was the distal region of tfpi- promoter where the point mutation located was not critical for tfpi- promoter activity. the deletion and scan mutation of tfpi- promoter support this idea. cancers often exhibit aberrant methylations at gene promoter regions that is associated with loss of gene function. tfpi- promoter exhibits typical features of a housekeeping gene. analysis with methprimer revealed that two complete cpg islands spanning promoter and exon and. cpg island hypermethylation has been described in almost every tumor type including breast cancer. it had also been reported that dnmt inhibitor restored expression of tfpi- in several cancer cell lines. methylation at the 5' position of cytosine has been reported to alter or interfere with the correct binding of transcription factors to target sequences overlapping cpg dinucleotides, and it also recruit methyl-cpg binding activities that associate with histone deacetylases and other chromtin-modifying elements that lead to a transcriptionally silenced state. klf a candidate tumor suppressor gene in prostate cancer, contains aa and is present in many tissues, including placenta, heart, lung, liver and pancreas. as an activator of transcription, klf interacts with the core promoter element of a tata box-less gene and cooperate with other transcription factors in kruppel-like family, including sp and klf. it had already been well documented that the promoter region of tfpi- has a high gc-rich content, with the presence of typical gc boxes known as binding sites for the transcription factor sp. after we found that there were no difference of klf binding between mda-mb- and mcf- cell, we considered that the cooperation of klf and sp might be affected by abnormal hypermethylation in klf binding site. treating the cell line mda-mb- with trichostatin a which is a histone deacetylase inhibitor alone could not reactivate expression of tfpi- but tsa could enhance the effects of methylase inhibitor aza. the tsa insensitive component of dnmt repression may be mediated by its interaction with dnmt associated protein. these data indicated a cooperative model for histone deacetylation and dna methylation in maintaining the silence of tfpi- in high invasive breast cancer cells. another question needed to be addressed is why the sp binding site in tfpi- promoter remains unmethylated in high metastasis breast cancer cell. a possible proposal is that sp elements protect a cpg island from de novo methylation, occurred in tumor genesis and this needs to be further investigated.  in the present work, we shown that: 1) expression of tfpi- was repressed in highly invasive breast cancer cell lines; 2) the - ~ - region was critical for tfpi- promoter activity and a klf binding site existed in this region which was abnormally hypermethylated in mda-mb-435; 3) the cpg methylation in the binding site of klf- blocked the binding of klf to tfpi- promoter, diminished the trans-activation of klf on the expression of tfpi- gene. on the basis of these findings, we conclude that the down regulation of tfpi- in highly metastasis breast cancer was due to the abnormal hypermethylation in cpg dinucleotides of klf- binding site. 
we found that both protein and mrna of tfpi- could not be detected in highly invasive breast cancer cell line mda-mb- to further investigate the mechanism of tfpi- repression in breast cancer cells, kb tfpi- 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. here, we have investigated the mechanism of dna methylation on the repression of tfpi- in breast cancer cell lines. scan mutation and informatics analysis identified a potential klf binding site in tfpi- promoter. in this study, we found that the cpg islands in tfpi- promoter was hypermethylated in highly invasive breast cancer cell line, and dna methylation in the entire promoter region caused tfpi- repression by inducing inactive chromatin structure and decreasing klf binding to its dna binding sequence. it was revealed, by bisulfite modified sequence, that the cpg island in tfpi- promoter region was hypermethylated in mda-mb- finally, using emsa and chip assay, we demonstrated that the cpg methylation in the binding site of klf- diminished the binding of klf to tfpi- promoter.
 tissue factor pathway inhibitor- 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- in breast cancer cell lines.  we found that both protein and mrna of tfpi- could not be detected in highly invasive breast cancer cell line mda-mb- to further investigate the mechanism of tfpi- repression in breast cancer cells, kb tfpi- 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 klf binding site in tfpi- promoter. it was revealed, by bisulfite modified sequence, that the cpg island in tfpi- promoter region was hypermethylated in mda-mb- finally, using emsa and chip assay, we demonstrated that the cpg methylation in the binding site of klf- diminished the binding of klf to tfpi- promoter.  in this study, we found that the cpg islands in tfpi- promoter was hypermethylated in highly invasive breast cancer cell line, and dna methylation in the entire promoter region caused tfpi- repression by inducing inactive chromatin structure and decreasing klf binding to its dna binding sequence. 