BACKGROUND
prostate cancer is the most frequently diagnosed neoplasia in men and one of the leading causes of cancer-related deaths in men over  <dig>  <cit> .
although early prostate specific antigen  detection and surgery have decreased the death rate, most of the patients still die of metastasis and recurrence of prostate cancer  <cit> .
however, the mechanisms involved in the onset and progression of prostate cancer are poorly understood at the molecular level.
it is important to understand the molecular biology of this cancer for its prevention, early diagnosis, and effective treatment.
pcan <dig>  is highly expressed in prostate epithelial tissue and frequently mutated in prostate tumors  <cit> .
pcan <dig> expression is undetectable in the highly undifferentiated du <dig> and pc- <dig> prostate cancer cell lines and weakly detected in the more differentiated lncap cell line  <cit> .
this gene is localized to chromosome 4q <dig>  a region of the genome that experiences frequent loss of heterozygosity  in prostate cancer.
it is mutated in 35% of the tumor samples  <cit> .
therefore,pcan <dig> has been proposed to have tumor suppressing function in prostate cancer.
nkx <dig>  is a prostate-specific homeobox gene that is thought to play an important role in the normal development of prostate and carcinogenesis.
in mice nkx <dig>  is exclusively expressed in prostate epithelium  <cit> .
its targeted disruption leads to aberrations in prostate ductal morphogenesis and secretory protein production, and epithelial hyperplasia and dysplasia  <cit> .
notably nkx <dig>  mutant mice display the pathologic changes of prostatic intraepithelial neoplasia   <cit>  that is the presumed precursor to prostate cancer in humans, implying that loss of nkx <dig>  expression correlates with the initiation of prostate carcinogenesis.
human nkx <dig>  has been mapped to human chromosome 8p <dig>  a region with frequent loss of heterozygosity in human prostate cancer  <cit> .
this gene has been proposed to have tumor suppressing function  <cit> .
it also inhibits the growth of cultured prostate cancer cells  <cit>  in vitro.
the strong association of nkx <dig>  with prostate development and prostate cancer makes this gene an attractive molecular target for further study.
nkx <dig>  and pcan <dig> are both prostate-specific genes strongly related to prostate development and prostate cancer.
studying the regulatory mechanisms of their expression is important for understanding their roles in prostate development and prostate cancer.
in this study, we cloned and characterized a  <dig>  kb fragment upstream of the pcan <dig> gene.
analysis of the  <dig>  kb sequence with matinspector  <dig>  revealed potential binding sites of some important transcription factors, including nkx <dig> , p <dig>  sp <dig>  cebp and ppar/rxr heterodimers.
in our study, the eukaryotic expression plasmids containing nkx <dig> , p <dig>  sp <dig>  cebpα and pparγ were respectively used to study their effects on pcan <dig> expression.
we found that nkx <dig>  upregulated pcan <dig> promoter activity and mrna expression.
to explore the regulatory mechanisms of nkx <dig>  on pcan <dig> transcription, we focused on identifying the functional nkx <dig>  binding sites  upstream of the pcan <dig> gene.
we demonstrated that nkx <dig>  upregulated pcan <dig> gene transcription through direct binding with nbss upstream of the pcan <dig> gene.
our study provided a molecular mechanism for the regulation of pcan <dig> gene expression.
RESULTS
effects of nkx <dig>  on pcan <dig> promoter activity and mrna expression
in our previous work, a  <dig>  kb promoter fragment  of the pcan <dig> gene amplified by pcr was inserted into pgl3-basic vector to form the pcan <dig> promoter-luciferase reporter plasmid designated as pgl3-ppcan <dig>  firefly luciferase expression driven by the  <dig>  kb pcan <dig> promoter was used to evaluate the promoter activity  <cit> .
to detect the effects of nkx <dig>  on pcan <dig> gene expression, lncap and pc- <dig> cells were harvested  <dig> h after cotransfection with pgl3-ppcan <dig> and pcdna <dig> -nkx <dig> .
the control cells were cotransfected with pgl3-ppcan <dig> and pcdna <dig>   plasmid.
the pcan <dig> promoter activity detected by dual-luciferase reporter assays was enhanced by  <dig> -fold in lncap cells and by  <dig> -fold in pc- <dig> cells with nkx <dig>  cotransfection, compared with pcan <dig> promoter activity in the control cells .
the pcan <dig> mrna expression level in lncap cells, as detected by rt-pcr, was increased significantly by nkx <dig>  cotransfection, compared with that of the control cells transfected with pcdna <dig>   plasmid .
the pcan <dig> mrna expression level in pc- <dig> cells was undetectable by rt-pcr .
the expression of endogenous nkx <dig>  in lncap cells was knocked down by rnai, which made the pcan <dig> mrna expression decrease, as detected by rt-pcr .
identification of specific nkx <dig>  binding sites  with nuclear extracts
analysis of the  <dig>  kb promoter sequence with matinspector  <dig>  revealed five potential nkx <dig>  transcription factor binding sites .
they were located at - <dig> to - <dig> , - <dig> to  <dig> , - <dig> to - <dig> , - <dig> to  <dig>  and - <dig> to - <dig> , upstream of the pcan <dig> gene.
to investigate the binding activities of these five nbss with nkx <dig>  transcription factor, we carried out electrophoretic mobility shift assays .
it was performed with nkx <dig> -transfected nuclear extracts from lncap cells and synthesized oligonucleotide probes containing these five nbs sequences.
the results showed that dna-protein binding complexes were identified for the nbs <dig> probe and the nbs <dig> probe .
the bindings of nbs <dig> and nbs <dig> with nuclear extracts proved to be specific, as they were blocked by a 250-fold excess of unlabeled nbs <dig> probe  or nbs <dig> probe  and by anti-nkx <dig>  antibody , but not by unlabeled mutant nbs <dig> probe or mutant nbs <dig> probe .
nkx <dig>  binds to nbs <dig> and nbs <dig> upstream of the pcan <dig> gene in living cells
to determine whether nkx <dig>  also binds to the nbss in vivo, we performed chromatin immunoprecipitation  assays, which are used to define interactions of proteins with specific dna elements in living cells.
chip was carried out in lncap cells transfected with pcdna <dig> -nkx <dig> .
after cross-linking with formaldehyde, cell lysates were immunoprecipitated with anti-nkx <dig>  antibody or rabbit igg .
the dna purified from this coprecipitation was analyzed by pcr with primers  spanning the nbss in the pcan <dig> promoter.
as shown in fig.
<dig>  we observed a clear pcr product using nbs <dig> primer or nbs <dig> primer but no band was observed using nbs <dig> primer or nbs <dig>  primer, and no pcr product was identified for all primers with rabbit igg precipitation complexes, indicating that nkx <dig>  bound to nbs <dig> and nbs <dig> upstream of the pcan <dig> gene in living cells.
nbs <dig> f and r span nbs1;
nbs <dig> f and r span nbs2; nbs <dig> f and r span nbs3; bns <dig>  f and r span nbs <dig> and nbs5
interaction of nkx <dig>  with nbs <dig> or nbs <dig> stimulates luciferase reporter expression driven by sv <dig> promoter
to investigate the interactions between nkx <dig>  and these five potential nbss in vivo, five pgl3-nbs-promoter luciferase plasmids were constructed  and cotransfected with pcdna <dig> -nkx <dig>  plasmid respectively into lncap cells, while the control cells were cotransfected with pcdna <dig>   vector.
the luciferase reporter assays showed that when cotransfected with nkx <dig>  expression plasmid, nbs <dig> and nbs <dig> enhanced sv <dig> promoter activity by  <dig> -fold and  <dig> -fold, respectively, compared with that of the control cells that were cotransfected with pcdna <dig>   vector.
nbs <dig>  nbs <dig>  nbs <dig> showed no significant effects on sv <dig> promoter activity.
these results suggested that nbs <dig> and nbs <dig> were the functional cis-elements in vivo for the upregulation by nkx <dig> .
deletion of nbs <dig> and nbs <dig> in the pcan <dig> promoter abolishes the positive regulation by nkx <dig>
the binding assays of five nbss in vivo and in vitro suggested that nbs <dig> and nbs <dig> in the pcan <dig> promoter were involved in the positive regulation of pcan <dig> expression by nkx <dig> .
to further confirm this observation, the sequences of nbs <dig>  or both nbs <dig> and nbs <dig> were deleted from pgl3-ppcan <dig> to examine the effects of the deletions on pcan <dig> promoter activity.
the results in fig.
<dig> showed that deletion of nbs <dig>  or both nbs <dig> and nbs <dig>  reduced the promoter activity to 75% or 50% of the wild-type promoter .
with cotransfection of the nkx <dig>  expression plasmid, deletion of nbs <dig> partially abolished, and deletion of both nbs <dig> and nbs <dig> completely abolished nkx <dig>  stimulation of the pcan <dig> promoter.
these findings suggested that nbs <dig> and nbs <dig> upstream of the pcan <dig> gene were functional cis-elements mediating the positive regulation by nkx <dig>  of pcan <dig> gene transcription.
discussion
nkx <dig>  is a prostate-specific homeobox gene that is thought to play important roles in normal prostate development.
nkx <dig>  protein has been proposed to act as a specific tumor suppressor in prostate.
loss of nkx <dig>  expression correlates with prostate carcinogenesis  <cit>  and prostate tumor progression  <cit> .
it was reported that nkx <dig>  could collaborate with other transcription factors, such as serum response factor   <cit> , sp-family protein  <cit>  and prostate derived ets factor   <cit> , to regulate the expression of target genes.
the potential for nkx <dig>  to exert a differentiating and growth suppressing effect on prostatic epithelium was confirmed by targeted gene disruption of nkx <dig>  in mice  <cit> .
deletion of either one or both copies of nkx <dig>  resulted in prostatic epithelial hyperplasia and dysplasia that increased in severity with age.
magee et al.
<cit>  has analyzed the expression profiles of prostate tissues from wild-type,nkx <dig> +/-, and nkx <dig> -/- mice and identified nkx <dig>  target genes.
however, the genes directly regulated by human nkx <dig>  have yet to be identified.
in the present study, we found that nkx <dig>  upregulated the expression of the pcan <dig> gene in lncap prostate cancer cells and identified two functional nkx <dig>  binding sites upstream of the pcan <dig> gene.
pcan <dig> is highly expressed in prostate epithelial tissue and was initially identified in a screen for prostate-specific genes.
as an important gene in prostate cancer initiation or progression  <cit> , it has been shown that pcan <dig> is frequently mutated or deleted in prostate tumor samples  <cit>  and differentially expressed in tumor versus normal prostate tissue, demonstrating a prostate tumor suppressor role  <cit> .
so far, little is known about the regulatory mechanisms of pcan <dig> gene expression as well as the relevant regulatory elements and factors.
cross et al.
<cit>  has identified pcan <dig> gene in prostate epithelial tissue and demonstrated its expression pattern in different cells.
they did an initial characterization of  <dig>  kb sequence upstream of the initiation sites with the matinspector program, in which they found several important transcription factor binding sites, including nkx <dig>  binding sites.
in our study, we cloned a  <dig>  kb pcan <dig> promoter sequence and five nkx <dig>  binding sites were found in this region with the matinspector .
we further performed reporter assays, rnai, emsa and chip to demonstrate that nkx <dig>  could directly bind to nkx <dig>  binding sites in the pcan <dig> promoter to enhance pcan <dig> gene expression in prostate cancer cells.
this finding provides a foundation for future studies of the regulatory mechanisms and roles of nkx <dig>  on pcan <dig> gene expression in prostate development and prostate cancer.
nkx <dig>  is proposed to be a nuclear transcriptional factor.
it preferentially binds the taagta sequence  <cit>  to regulate its target gene expression.
it is reported that nkx <dig>  acts as a transcription factor through recruitment of some corepressors  <cit>  or coactivators  <cit>  to repress or activate its target genes.
our study provided some evidences to show that nkx <dig>  directly binds to nkx <dig>  binding sites in the pcan <dig> promoter to activate pcan <dig> gene expression in prostate cancer cells.
however, further studies will be required to explore the regulatory mechanisms and the cofactors involved in the regulation.
nkx <dig>  is a prostate-specific gene required for maintenance of the normal differentiated state of the prostate epithelium.
it is proposed to have tumor suppressing function.
however, it is not a classic tumor suppressor gene.
despite that loss of function of nkx <dig>  predisposes to prostate cancer, it is not sufficient for tumorigenesis.
moreover, while one allele of nkx <dig>  is lost by means of chromosomal deletion in prostate cancer, the other allele does not undergo mutational inactivation.
these features are not consistent with activities of "classic" tumor suppressor genes.
instead, nkx <dig>  appears to act more like a tumor modulator, serving as a regulator of differentiation, which in turn prevents cancer initiation.
our finding that nkx <dig>  upregulates pcan <dig> expression will give a clue for further exploring the relationships between nkx <dig>  and pcan <dig> and understanding the functional importance of nxk <dig>  in regulating pcan <dig> in prostate differentiation and carcinogenesis.
identification of the specific biological functions of nkx <dig>  in prostate cancer may provide targets for the early diagnosis and prevention of prostate cancer.
nkx <dig>  is an androgen regulated gene and its expression is upregulated by androgens.
it is presumed that androgens upregulate pcan <dig> expression through increasing nkx <dig>  expression.
in the experiments we have analyzed the effect of androgen  on endogenous pcan <dig> and nkx <dig>  expression in lncap cells.
our results showed that r <dig>  increased nkx <dig>  expression but have no significant effects on pcan <dig> expression in rt-pcr  and pcan <dig> promoter activity in luciferase reporter assays .
so far, it is not very clear whether or not pcan <dig> gene expression is regulated directly by androgens.
it may be possible that pcan <dig> is negatively regulated by androgens and nkx <dig>  compensates this effect through positively regulating pcan <dig>  in addition, ar, nkx <dig>  and pcan <dig> play different roles in prostate development and cancer, and their interactions in prostate are very complicated and need further to be investigated.
CONCLUSIONS
in conclusion, we cloned and characterized the human pcan <dig> promoter, and identified two functional nkx <dig>  binding sites upstream of the pcan <dig> gene, which were involved in the positive regulation by nkx <dig>  of pcan <dig> gene expression.
both nkx <dig>  and pcan <dig> are related to prostate development and prostate cancer.
our findings will contribute to the understanding of molecular regulatory mechanisms of pcan <dig> gene expression in prostate development and cancer.

 prostate cancer is the most frequently diagnosed neoplasia in men and one of the leading causes of cancer-related deaths in men over. although early prostate specific antigen detection and surgery have decreased the death rate, most of the patients still die of metastasis and recurrence of prostate cancer. however, the mechanisms involved in the onset and progression of prostate cancer are poorly understood at the molecular level. it is important to understand the molecular biology of this cancer for its prevention, early diagnosis, and effective treatment. pcan is highly expressed in prostate epithelial tissue and frequently mutated in prostate tumors. pcan expression is undetectable in the highly undifferentiated du and pc- prostate cancer cell lines and weakly detected in the more differentiated lncap cell line. this gene is localized to chromosome 4q a region of the genome that experiences frequent loss of heterozygosity in prostate cancer. it is mutated in 35% of the tumor samples. therefore,pcan has been proposed to have tumor suppressing function in prostate cancer. nkx is a prostate-specific homeobox gene that is thought to play an important role in the normal development of prostate and carcinogenesis. in mice nkx is exclusively expressed in prostate epithelium. its targeted disruption leads to aberrations in prostate ductal morphogenesis and secretory protein production, and epithelial hyperplasia and dysplasia. notably nkx mutant mice display the pathologic changes of prostatic intraepithelial neoplasia that is the presumed precursor to prostate cancer in humans, implying that loss of nkx expression correlates with the initiation of prostate carcinogenesis. human nkx has been mapped to human chromosome 8p a region with frequent loss of heterozygosity in human prostate cancer. this gene has been proposed to have tumor suppressing function. it also inhibits the growth of cultured prostate cancer cells in vitro. the strong association of nkx with prostate development and prostate cancer makes this gene an attractive molecular target for further study. nkx and pcan are both prostate-specific genes strongly related to prostate development and prostate cancer. studying the regulatory mechanisms of their expression is important for understanding their roles in prostate development and prostate cancer. in this study, we cloned and characterized a kb fragment upstream of the pcan gene. analysis of the kb sequence with matinspector revealed potential binding sites of some important transcription factors, including nkx, p sp cebp and ppar/rxr heterodimers. in our study, the eukaryotic expression plasmids containing nkx, p sp cebpα and pparγ were respectively used to study their effects on pcan expression. we found that nkx upregulated pcan promoter activity and mrna expression. to explore the regulatory mechanisms of nkx on pcan transcription, we focused on identifying the functional nkx binding sites upstream of the pcan gene. we demonstrated that nkx upregulated pcan gene transcription through direct binding with nbss upstream of the pcan gene. our study provided a molecular mechanism for the regulation of pcan gene expression.  effects of nkx on pcan promoter activity and mrna expression in our previous work, a kb promoter fragment of the pcan gene amplified by pcr was inserted into pgl3-basic vector to form the pcan promoter-luciferase reporter plasmid designated as pgl3-ppcan firefly luciferase expression driven by the kb pcan promoter was used to evaluate the promoter activity. to detect the effects of nkx on pcan gene expression, lncap and pc- cells were harvested h after cotransfection with pgl3-ppcan and pcdna -nkx. the control cells were cotransfected with pgl3-ppcan and pcdna plasmid. the pcan promoter activity detected by dual-luciferase reporter assays was enhanced by -fold in lncap cells and by -fold in pc- cells with nkx cotransfection, compared with pcan promoter activity in the control cells. the pcan mrna expression level in lncap cells, as detected by rt-pcr, was increased significantly by nkx cotransfection, compared with that of the control cells transfected with pcdna plasmid. the pcan mrna expression level in pc- cells was undetectable by rt-pcr. the expression of endogenous nkx in lncap cells was knocked down by rnai, which made the pcan mrna expression decrease, as detected by rt-pcr. identification of specific nkx binding sites with nuclear extracts analysis of the kb promoter sequence with matinspector revealed five potential nkx transcription factor binding sites. they were located at - to -, - to, - to -, - to and - to -, upstream of the pcan gene. to investigate the binding activities of these five nbss with nkx transcription factor, we carried out electrophoretic mobility shift assays. it was performed with nkx -transfected nuclear extracts from lncap cells and synthesized oligonucleotide probes containing these five nbs sequences. the results showed that dna-protein binding complexes were identified for the nbs probe and the nbs probe. the bindings of nbs and nbs with nuclear extracts proved to be specific, as they were blocked by a 250-fold excess of unlabeled nbs probe or nbs probe and by anti-nkx antibody, but not by unlabeled mutant nbs probe or mutant nbs probe. nkx binds to nbs and nbs upstream of the pcan gene in living cells to determine whether nkx also binds to the nbss in vivo, we performed chromatin immunoprecipitation assays, which are used to define interactions of proteins with specific dna elements in living cells. chip was carried out in lncap cells transfected with pcdna -nkx. after cross-linking with formaldehyde, cell lysates were immunoprecipitated with anti-nkx antibody or rabbit igg. the dna purified from this coprecipitation was analyzed by pcr with primers spanning the nbss in the pcan promoter. as shown in fig. we observed a clear pcr product using nbs primer or nbs primer but no band was observed using nbs primer or nbs primer, and no pcr product was identified for all primers with rabbit igg precipitation complexes, indicating that nkx bound to nbs and nbs upstream of the pcan gene in living cells. nbs f and r span nbs1; nbs f and r span nbs2; nbs f and r span nbs3; bns f and r span nbs and nbs5 interaction of nkx with nbs or nbs stimulates luciferase reporter expression driven by sv promoter to investigate the interactions between nkx and these five potential nbss in vivo, five pgl3-nbs-promoter luciferase plasmids were constructed and cotransfected with pcdna -nkx plasmid respectively into lncap cells, while the control cells were cotransfected with pcdna vector. the luciferase reporter assays showed that when cotransfected with nkx expression plasmid, nbs and nbs enhanced sv promoter activity by -fold and -fold, respectively, compared with that of the control cells that were cotransfected with pcdna vector. nbs nbs nbs showed no significant effects on sv promoter activity. these results suggested that nbs and nbs were the functional cis-elements in vivo for the upregulation by nkx. deletion of nbs and nbs in the pcan promoter abolishes the positive regulation by nkx the binding assays of five nbss in vivo and in vitro suggested that nbs and nbs in the pcan promoter were involved in the positive regulation of pcan expression by nkx. to further confirm this observation, the sequences of nbs or both nbs and nbs were deleted from pgl3-ppcan to examine the effects of the deletions on pcan promoter activity. the results in fig. showed that deletion of nbs or both nbs and nbs reduced the promoter activity to 75% or 50% of the wild-type promoter. with cotransfection of the nkx expression plasmid, deletion of nbs partially abolished, and deletion of both nbs and nbs completely abolished nkx stimulation of the pcan promoter. these findings suggested that nbs and nbs upstream of the pcan gene were functional cis-elements mediating the positive regulation by nkx of pcan gene transcription. discussion nkx is a prostate-specific homeobox gene that is thought to play important roles in normal prostate development. nkx protein has been proposed to act as a specific tumor suppressor in prostate. loss of nkx expression correlates with prostate carcinogenesis and prostate tumor progression. it was reported that nkx could collaborate with other transcription factors, such as serum response factor, sp-family protein and prostate derived ets factor, to regulate the expression of target genes. the potential for nkx to exert a differentiating and growth suppressing effect on prostatic epithelium was confirmed by targeted gene disruption of nkx in mice. deletion of either one or both copies of nkx resulted in prostatic epithelial hyperplasia and dysplasia that increased in severity with age. magee et al. has analyzed the expression profiles of prostate tissues from wild-type,nkx +/-, and nkx -/- mice and identified nkx target genes. however, the genes directly regulated by human nkx have yet to be identified. in the present study, we found that nkx upregulated the expression of the pcan gene in lncap prostate cancer cells and identified two functional nkx binding sites upstream of the pcan gene. pcan is highly expressed in prostate epithelial tissue and was initially identified in a screen for prostate-specific genes. as an important gene in prostate cancer initiation or progression, it has been shown that pcan is frequently mutated or deleted in prostate tumor samples and differentially expressed in tumor versus normal prostate tissue, demonstrating a prostate tumor suppressor role. so far, little is known about the regulatory mechanisms of pcan gene expression as well as the relevant regulatory elements and factors. cross et al. has identified pcan gene in prostate epithelial tissue and demonstrated its expression pattern in different cells. they did an initial characterization of kb sequence upstream of the initiation sites with the matinspector program, in which they found several important transcription factor binding sites, including nkx binding sites. in our study, we cloned a kb pcan promoter sequence and five nkx binding sites were found in this region with the matinspector. we further performed reporter assays, rnai, emsa and chip to demonstrate that nkx could directly bind to nkx binding sites in the pcan promoter to enhance pcan gene expression in prostate cancer cells. this finding provides a foundation for future studies of the regulatory mechanisms and roles of nkx on pcan gene expression in prostate development and prostate cancer. nkx is proposed to be a nuclear transcriptional factor. it preferentially binds the taagta sequence to regulate its target gene expression. it is reported that nkx acts as a transcription factor through recruitment of some corepressors or coactivators to repress or activate its target genes. our study provided some evidences to show that nkx directly binds to nkx binding sites in the pcan promoter to activate pcan gene expression in prostate cancer cells. however, further studies will be required to explore the regulatory mechanisms and the cofactors involved in the regulation. nkx is a prostate-specific gene required for maintenance of the normal differentiated state of the prostate epithelium. it is proposed to have tumor suppressing function. however, it is not a classic tumor suppressor gene. despite that loss of function of nkx predisposes to prostate cancer, it is not sufficient for tumorigenesis. moreover, while one allele of nkx is lost by means of chromosomal deletion in prostate cancer, the other allele does not undergo mutational inactivation. these features are not consistent with activities of "classic" tumor suppressor genes. instead, nkx appears to act more like a tumor modulator, serving as a regulator of differentiation, which in turn prevents cancer initiation. our finding that nkx upregulates pcan expression will give a clue for further exploring the relationships between nkx and pcan and understanding the functional importance of nxk in regulating pcan in prostate differentiation and carcinogenesis. identification of the specific biological functions of nkx in prostate cancer may provide targets for the early diagnosis and prevention of prostate cancer. nkx is an androgen regulated gene and its expression is upregulated by androgens. it is presumed that androgens upregulate pcan expression through increasing nkx expression. in the experiments we have analyzed the effect of androgen on endogenous pcan and nkx expression in lncap cells. our results showed that r increased nkx expression but have no significant effects on pcan expression in rt-pcr and pcan promoter activity in luciferase reporter assays. so far, it is not very clear whether or not pcan gene expression is regulated directly by androgens. it may be possible that pcan is negatively regulated by androgens and nkx compensates this effect through positively regulating pcan in addition, ar, nkx and pcan play different roles in prostate development and cancer, and their interactions in prostate are very complicated and need further to be investigated.  in conclusion, we cloned and characterized the human pcan promoter, and identified two functional nkx binding sites upstream of the pcan gene, which were involved in the positive regulation by nkx of pcan gene expression. both nkx and pcan are related to prostate development and prostate cancer. our findings will contribute to the understanding of molecular regulatory mechanisms of pcan gene expression in prostate development and cancer. 
nkx and pcan are both prostate-specific genes related to prostate development and prostate cancer. in the present study, we found that nkx upregulated pcan gene transcription in lncap prostate cancer cells. luciferase reporter assays, electrophoretic mobility shift assays, chromatin immunoprecipitation and rna interference were performed to study the effects of nkx on pcan gene expression in prostate cancer cells. our results showed that pcan promoter activity and mrna expression were increased by transfection with the nkx containing plasmid and that pcan mrna expression was decreased by rna interference targeting human nkx in lncap prostate cancer cells. the results of electrophoretic mobility shift assays and chromatin immunoprecipitation showed that nkx bound to nbs and nbs upstream of the pcan gene. our results suggested that two functional nkx binding sites located at - to - and - to - upstream of the pcan gene were involved in the positive regulation of pcan gene transcription by nkx. the luciferase reporter assays showed that nbs and nbs enhanced the promoter activity in pgl3-promoter vector with cotransfection of the nkx containing plasmid. so far, little is known about the regulatory mechanisms of the expression of these two genes. to understand the regulatory mechanisms, our work focused on identifying the functional nkx binding sites upstream of the pcan gene, which might be involved in the positive regulation of pcan expression by nkx. furthermore, the deletion of nbs or both nbs and nbs reduced pcan promoter activity and abolished the positive regulation of pcan expression by nkx.
 nkx and pcan are both prostate-specific genes related to prostate development and prostate cancer. so far, little is known about the regulatory mechanisms of the expression of these two genes. in the present study, we found that nkx upregulated pcan gene transcription in lncap prostate cancer cells. to understand the regulatory mechanisms, our work focused on identifying the functional nkx binding sites upstream of the pcan gene, which might be involved in the positive regulation of pcan expression by nkx.  we cloned and characterized a kb fragment upstream of the pcan gene. analysis of the kb sequence with matinspector revealed five potential binding sites of nkx transcription factor. luciferase reporter assays, electrophoretic mobility shift assays, chromatin immunoprecipitation and rna interference were performed to study the effects of nkx on pcan gene expression in prostate cancer cells. our results showed that pcan promoter activity and mrna expression were increased by transfection with the nkx containing plasmid and that pcan mrna expression was decreased by rna interference targeting human nkx in lncap prostate cancer cells. the results of electrophoretic mobility shift assays and chromatin immunoprecipitation showed that nkx bound to nbs and nbs upstream of the pcan gene. the luciferase reporter assays showed that nbs and nbs enhanced the promoter activity in pgl3-promoter vector with cotransfection of the nkx containing plasmid. furthermore, the deletion of nbs or both nbs and nbs reduced pcan promoter activity and abolished the positive regulation of pcan expression by nkx.  our results suggested that two functional nkx binding sites located at - to - and - to - upstream of the pcan gene were involved in the positive regulation of pcan gene transcription by nkx. 