BACKGROUND
decoy receptor  <dig>  is a member of the tumor necrosis factor receptor  superfamily.
it has been shown to be the decoy receptor for fas ligand , light and tl1a  <cit> , also known as tr <dig>  <cit> .
dcr <dig> is mostly expressed in tumor cells and competitively inhibits tnf signaling.
overexpression of dcr <dig> in tumor cells protects them from apoptosis.
dcr <dig> protects tumor cells from immune surveillance as it contributes to the suppression of the host anti-tumor immunity.
dcr <dig> mrna and protein are amplified in various malignant tissues, such as lung cancer, colon cancer, gastric cancer, oesophageal carcinoma, pancreas cancer and malignant melanoma  <cit> .
wu et al.
<cit>  reported that dcr <dig> could not be detected in non-tumor patients, but could be detected in  <dig> %  of patients with malignant cancers.
this phenomenon demonstrates that the elevated dcr <dig> expression is significantly correlated with tumorigenesis and tumor progression.
wu et al.
<cit>  reported that dcr <dig> was highly expressed in human gastric cancer , and positively correlated with the development and metastases of gastric lesions.
gastric cancer patients with a high dcr <dig> expression presented a more advanced pn2- <dig> disease than those with a low dcr <dig> expression.
the dcr <dig> for fasl may be involved in the progression of gastric cancer.
further evaluation of the possible roles of dcr <dig> and the regulation of dcr <dig> expression in malignant cells is very important for the development of new strategies for controlling the growth of malignant cells that escape the host immune surveillance.
at the early stage, the mapk/erk pathway is activated in tumors, which is a notable sign for many kinds of cancers in humans.
recently, several lines of evidence suggested that erk could be a parameter for predicting the prognosis of various cancers such as breast cancer, colon cancer, pancreas cancer and cholangiocarcinoma.
wang et al.
<cit>  reported that the expression of erk1/ <dig> was high in cholangiocarcinoma, which correlated with the tnm stages.
kim et al.
<cit>  found that lps induced dcr <dig> release in human intestinal epithelial cells , which appeared to be via the activation of mitogen-activated protein kinases , such as extracellular signal-regulated kinase  <dig> and  <dig>  and c-jun nh2-terminal protein kinase .
lps-induced dcr <dig> release in sw <dig> cells was abolished by erk1/ <dig> and jnk inhibitors.
furthermore, yang et al.
<cit>  reported that 3 g/ml dcr <dig> markedly induced the phosphorylation of erk and p <dig>
in accordance with these reports, we propose that dcr <dig> and erk1/ <dig> are closely related.
the dcr <dig> genes closely correlated with the occurrence and development of many kinds of tumors.
in this study we investigated the expression level and location of dcr <dig> and erk1/ <dig> in gastric cancer patients of different ages, gender, stage and differentiation, to explore the relationship between dcr3/erk1/ <dig> and gastric cancer occurrence and development.
furthermore, we provide suggestions for clinical diagnosis of gastric cancers.
methods
clinical samples
tumors were from gastric cancer patients who were undergone endoscopic biopsy or curative operations in zhongshan hospital affiliated with xiamen university.
tumor tissues from which dna and protein were isolated were from fresh specimen of resection surgery.
all samples were obtained with patient consent and approval of the committee on medical ethics of zhongshan hospital xiamen university.
mice
all the experiments were performed using 6–8 weeks male nude mice purchased from model animal research center of medical college xiamen university.
all the animals were housed under specific pathogen free conditions with constant access to water and chow.
all experimental procedures were carried out following the approval of the animal care and use committee of xiamen university.
cell culture
the human gastric cancer cell line bgc <dig> was maintained in our laboratory, which was cultured in flasks with dulbecco’s modified eagle’s medium  supplemented with 10% fetal bovine serum , and 1% penicillin-streptomycin at 37°c in an humidified atmosphere of 5% co <dig>
reagents
dmem, fbs and penicillin-streptomycin were purchased from hyclone corporation .
hematoxylin-and-eosin  assay kit were purchased from chemicon international, inc .
erk1/ <dig> and dcr <dig> abs were purchased from santa cruz bio technology.
rt-rcr reagents were purchased from takara .
the primer sequence was synthesized from sangon .
in vivo animal tumor models
tumors were generated in male nude mice by intramuscular  injection of bgc <dig> cells  into the right flank.
tumor measurements were converted to tumor volume  by the formula , where l and w are the length and width, respectively.
measurements were made with a vernier caliper.
all tumor-bearing mice were divided randomly into groups .
rt-pcr and western blotting analysis for examining the expression of dcr3/erk
total rna of dcr <dig> and erk1/ <dig> was extracted from stimulated cells using trizol.
to measure erk/dcr <dig> gene copy number, dna from fresh tumor samples was analyzed with rt-pcr.
the upstream and downstream primers for erk <dig> mrna were 5'-cctgctcatcaacaccacc-3’ and 5'-cgtagccacatactccgtca-3’, and for erk <dig> mrna were 5'-tcttcc agccctccttcctg-3' and 5'-cgtttctgcgccgttaggt-3'.
the samples were denatured at 95°c for 4 min, followed by  <dig> cycles of amplification .
the products were 300-bp and 400-bp fragments respectively.
for dcr <dig> mrna, the upstream primer was 5'- gcaaagccaaggattccccctg -3’ and the downstream primer was 5'-ggcactgctctgagctggagctg-3’.
the samples were denatured at 95°c for 4 min, followed by  <dig> cycles of amplification .
the product was a 921-bp fragment.
bgc <dig> cells were harvested and lysed in cell lysis buffer  nonidet p- <dig>  150 mm nacl, 50 mm tris–hcl , 1 mm pmsf, 2 g/ml aprotinin, and 2 g/ml leupeptin) which could release dcr <dig> and erk1/ <dig> proteins.
twenty-five micrograms of total lysate was fractionated by sds-page and subjected to western blotting analysis using the anti-erk or anti-dcr <dig> mab .
western blotting and elisa assay for examining the expression of dcr3/erk after inhibitors treatment
bgc <dig> cells culture supernatants were collected at various intervals, and levels of u <dig>  pd <dig>  apdc, mek1/ <dig> and erk1/ <dig> interferences were quantified using commercial elisa kits, according to the vendor’s instructions.
cells treated with erk1/ <dig> shrna , u0126/pd <dig>  and apdc , respectively.
after a 5- or 7-day incubation, cells were subjected to the cytokines as indicated assay.
twenty-five micrograms of total lysate was fractionated by sds-page and subjected to western blotting analysis using the anti-erk or anti-dcr <dig> mab .
immunohistochemistry analysis for the expression of dcr <dig> and erk1/2
tumor tissues were fixed in a formaldehyde medium and embedded in paraffin.
sections  <dig> mm thick were mounted on glass slides pretreated with  <dig> % poly-l-lysine.
they were then deparaffinaged in xy-lene, dehydrated in graded ethanol and soaked in 3% h2o <dig> for 10 min to eliminate endogenous peroxidase activity.
next, the slides were submerged in citrate buffer  and boiled at 92–98°c in a microwave oven for 10 min.
subsequently, they were rinsed  <dig> times with pbs for 10 min each and blocked with 10% normal goat serum in pbs for 1 hr at room temperature.
the slides were then reacted with the affinity-purified rabbit anti-tr <dig> ab  at room temperature for 2 hr.
after washing, the slides were incubated with biotinylated goat-anti-rabbit antibody for 10 min.
tr <dig> signal was revealed by streptavidin-peroxidase using dab as a substrate according to instructions from the histostain-plus kit .
dcr <dig> signals were revealed in brown.
finally, the slides were counterstained with hematoxylin and sealed with aqueous mounting media .
statistical analysis
data were presented as mean ± sd.
the significance of the difference between the groups was assessed by student`s two-tailed t-test.
probability value of less than  <dig>  was considered significant.
all means were calculated from at least three independent experiments.
RESULTS
cancer patients have elevated dcr <dig> levels
to study the correlation between dcr <dig> expression and tumor occurrence and development, tumors from  <dig> gastric cancer patients were collected and tested for dcr <dig> mrna and protein levels.
as shown in figure 1a, the 921 bp dcr <dig> bands were generated by rt-pcr from the tumor tissues of most patients , compared with the non-cancerous tissues  from the same organs of these patients.
these results demonstrate that dcr <dig> levels were significantly increased .
to further confirm our results, the dcr <dig> protein levels were examined by western blotting.
the results demonstrate that dcr <dig> protein could be detected in most cancer patients; dcr <dig> protein was detected in 74%  of the tumor tissues, but only in 6%  of the non-cancerous tissues .
expression of erk1/ <dig> in clinical samples
to identify the signaling molecules related to dcr <dig>  the expression levels of erk1/ <dig>  jnk and p <dig> were tested.
we found that only erk1/ <dig> could be detected in tumor samples .
erk <dig> mrna was detected in 74% of the samples , and erk <dig> mrna was detected in 64% of the samples .
at the protein level, erk <dig> was detected in 84%  and erk <dig> in 74%  of the tumor tissues .
these results indicate that patients with gastric cancer tumors have a higher erk1/2-positive occurrence compared with the non-cancerous tissues .
there was a positive correlation between the mrna and protein levels, as in  <dig> of  <dig> tumor tissues the erk <dig> protein level was elevated, of which  <dig> cases were consistent with the mrna expression.
in contrast, erk <dig> protein expression was detected only in five normal tissues.
when examining erk <dig>   <dig> of  <dig> cases of tumor tissues were positive for erk <dig> protein expression, of which  <dig> cases were consistent with the erk <dig> mrna expression, while only ten cases showed positive protein expression in normal tissues.
the eight randomly picked samples are shown in figure  <dig>
location of erk1/ <dig> in gastric cancer patients
to examine erk1/ <dig> distribution, tumors from  <dig> patients were tested by immunohistochemistry.
the results show that erk1/ <dig> were expressed in tumor tissues from most of the patients.
erk <dig> expression was found in the cytoplasm.
erk <dig> positive expression was found in some tumor cells .
dcr <dig> and erk1/ <dig> levels correlate with tumor invasion but not with age, gender or differentiation
the positive incidence of dcr <dig> mrna in tumor tissues of gastric cancer patients was  <dig> % , and of dcr <dig> protein  <dig> % , which was significantly higher than in non-cancerous tissues showing only 4%  and 6% , respectively .
the positive occurrence of erk1/ <dig> mrna expression was  <dig> %  and  <dig> % , and of protein expression  <dig> %  and  <dig> % .
both of them were significantly higher than the non-cancerous tissues, showing  <dig> %  and  <dig> % , and  <dig> %  and  <dig> % , respectively .
these results suggest that dcr <dig> and erk1/ <dig> expression levels correlated with tumor occurrence and development.
as shown in tables  <dig>   <dig> and  <dig>  there was no significant difference between tumor tissues from different ages, gender groups and patients with different differentiation stages of gastric cancer .
however, dcr <dig> and erk1/ <dig> expression levels were significantly high in tnm stage ii-iv .
thus, the expressions of dcr <dig> and erk1/ <dig> correlated with tumor invasion and tnm stage, but not with age, gender or differentiation.
expression levels of dcr <dig> and erk1/ <dig> are amplified in animal models
as shown in table  <dig>  after injecting bgc <dig> cells into the right flank of nude mice, the tumors grew each day.
rt-pcr was used to test dcr <dig> and erk1/ <dig> mrna levels in the tumors and western blot analysis was used to examine protein levels.
in gastric cancer animal models, dcr <dig>  erk <dig> and erk <dig> were detected at 921 bp, 300 bp and 400 bp, respectively.
in these tumor tissues dcr <dig> mrna was detected from day six, peaked on day  <dig>  and remained detected on day  <dig>  dcr <dig> was also detected from day six to day  <dig> in liver, while it was only detected on day  <dig> in heart and lung .
erk1/ <dig> mrna expressions were detected from day four in tumor tissues, and erk <dig> mrna peaked on day  <dig> .
note: △p <  <dig>  vs normal; **p <  <dig>  vs model.
dcr <dig> protein was detected on day six in tumor tissues and liver, and the expression remained until day  <dig>  dcr <dig> protein was detected on day  <dig> in spleen and by day  <dig> in heart, kidney and lung.
erk <dig> protein was detected on day four in tumor tissues, and continued to increase each day.
erk <dig> protein remained at a stable level in heart, liver and kidney, but it decreased in lung and spleen on day  <dig>  after reaching the peak.
erk <dig> was detected on day two in spleen and tumor tissues.
from day four, erk <dig> protein was detected in all six samples, and continued to increase till day  <dig>  these results suggest that erk <dig> and erk <dig> might have different effects on tumor occurrence, development and clonal expansion.
dcr <dig> expression decreased after inhibiting the expression or phosphorylation of erk1/ <dig> in bgc <dig> cells
to investigate the effect of erk1/ <dig> expression and phosphorylation on dcr <dig> expression, bgc <dig> cells were treated with erk1/ <dig> shrna or with inhibitors that specifically regulate the erk pathway.
western blot analysis confirmed that the inhibitors efficiently blocked the phosphorylation of the mek/erk pathway molecules, and that the shrna significantly reduced the expression of erk1/ <dig> .
as shown in figure 5a, when bgc <dig> cells were treated with erk1/ <dig> shrna, erk1/ <dig> and p-erk1/ <dig> levels declined compared with the control.
u <dig> is a very effective mek inhibitor, resulting in the inhibition of erk phosphorylation, as does pd <dig>  erk phosphorylation gradually declined as the concentrations of the drugs increased, although total erk1/ <dig> protein expression hardly changed .
apdc can inhibit nf-κb cell activation in a variety of cells.
through the degradation of iκb, apdc can decrease the translocation of nf-κb, thus blocking nf-κb activation.
as shown in figure  <dig> c, under different concentrations of apdc, changing the level of nf-κb inhibition can significantly attenuate erk1/ <dig> phosphorylation levels.
however, the specific mechanism requires further investigation.
to examine the effect of these inhibitors and shrna on dcr <dig> expression we used elisa analysis, which demonstrated that secreted dcr <dig> in the supernatant decreased after the different treatments .
statistical analysis showed that dcr <dig> secretion levels were significantly different between the experiment groups and control groups .
as shown in figure  <dig>  interference with erk1/ <dig> in bgc <dig> cells led to decreased dcr <dig> protein expression compared with the control group.
the trend matches the erk expression level in figure  <dig> and proves that the two are positively correlated.
furthermore, dcr <dig> and p-erk expression levels decreased when cells were treated with different concentrations of u <dig>  pd <dig> and apdc.
this data indicates that secretion of dcr <dig> positively correlated with p-erk1/ <dig> expression levels in bgc <dig> gastric cells.
it is worth noting that in the u <dig> group, dcr <dig> secretion levels increased when the drug concentration reached 40 μmol/l; however, the specific mechanism requires further investigation.
in the apdc group, dcr <dig> levels did not change significantly at concentrations higher than 20 μmol/l.
discussion
it has been demonstrated that the dcr <dig> gene is expressed at a low level in human embryo, lung, brain, liver, spleen, stomach, colon, lymph nodes and spinal cord, whereas it was expressed at a high level in cancers such as gastrointestinal cancer, hepatocellular carcinoma and pancreatic cancer  <cit> .
wu et al.
<cit>  reported that the expression of dcr <dig> in gastric cancer patients was significantly higher than normal.
dcr <dig> expression in the well-differentiated gastric cancer was significantly lower than that of poorly differentiated specimens .
the dcr <dig> expression level was significantly associated with lymph node metastasis and pathological stage, but did not correlate with tumor size, metastatic status, or histological types.
when patients were followed-up for 63 months, dcr <dig> overexpression was found to be associated with a significantly shortened survival rate  <cit> .
many reports have shown that high expression levels of erk1/ <dig> closely correlated with breast, colorectal and pancreatic cancer, as well as malignant melanoma, leukemia and myxoma  <cit> .
our research showed that in patients with gastric cancer, the positive incidence of dcr <dig> and erk1/ <dig> mrna was higher than that in the non-cancerous tissues .
rt-pcr and western blotting showed that the mrna and protein expression levels of dcr <dig> and erk1/ <dig> in tumor tissues were significantly higher than those in non-cancer tissues, suggesting that dcr <dig> and erk1/ <dig> levels correlate with tumor development but not with age, gender or differentiation .
our results showed that the positive incidence of expression of dcr <dig> and erk1/ <dig> mrna and dcr <dig> and erk1/ <dig> protein matched each other.
the immunohistochemistry results also demonstrated that in gastric tumors, erk1/ <dig> was highly expressed.
chen et al.
<cit>  reported that the positive rate of dcr <dig> expression was  <dig> %  in hepatocellular carcinoma, and that there was a significant correlation between dcr <dig> expression and metastasis as well as recurrence and differentiation.
in the mouse gastric model, rt-pcr showed that dcr <dig> mrna could be detected in tumors from day six.
erk1/ <dig> mrna and protein were also detected in tumors, and erk <dig> levels gradually increased in gastric tumors.
furthermore, they were also detected in heart, liver, spleen, lung and kidney of the gastric cancer animal model, suggesting that they have an important role in tumor progression.
erk1/ <dig> mrna was detected from day four in tumor tissues, and erk <dig> mrna peaked on day  <dig>  erk <dig> protein could also be detected on day four in tumor tissues, and continued to increase every day.
erk <dig> remained at a stable level in heart, liver and kidney, but it decreased in lung and spleen on day  <dig>  after reaching the peak.
erk <dig> was detected on day two in spleen and tumor tissues.
from day four, erk <dig> was detected in all six tissues, and continued to increase till day  <dig>  erk <dig> could not be detected in heart, lung and spleen by rt-pcr on day  <dig> in animal models, but protein levels could be detected.
these results suggest that erk <dig> and erk <dig> might have different effects on tumor occurrence, development and clonal expansion.
many studies indicated that the expression of erk1/ <dig> mrna and protein varies in different tumors and cells  <cit> .
some recent reports suggested they could be entirely opposite in some cases.
thus, the effects of erk <dig> and erk <dig> on the tumors are unlikely to be the same.
CONCLUSIONS
in conclusion, high expression of dcr <dig> and erk1/ <dig> may suppress tumor cell apoptosis and play an influential role in gastric cancer occurrence and development, which is an important mechanism in tumorigenesis  <cit> .
in our study, dcr <dig> and erk1/ <dig> presented an overexpression tendency, and participated in the tumor immunity.
we infer that in the tumor occurrence and developing process, the expression of erk1/ <dig> and dcr <dig> may be related to each other.
understanding the role of erk1/ <dig> in dcr <dig> expression might shed light on gastric cancer's diagnosis and identify a factor that regulates the expression of dcr <dig>  it might be a new marker for early diagnosis of gastric cancer.
dcr <dig> is a secreted protein, which can be detected in blood serum.
thus it could serve as a reliable index for clinical malignant tumor diagnosis, treatment and prognosis.
therefore, dcr <dig> has the potential of becoming a novel tumor marker in the future.
competing interest
the authors declare that they have no competing interest.
authors’ contributions
dy and xf carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript.
py, qw carried out the immunoassays.
fy, sy and bl participated in the sequence alignment.
zl participated in the design of the study and performed the statistical analysis.
gz conceived of the study, and participated in its design and coordination.
all authors read and approved the final manuscript.

 decoy receptor is a member of the tumor necrosis factor receptor superfamily. it has been shown to be the decoy receptor for fas ligand, light and tl1a, also known as tr. dcr is mostly expressed in tumor cells and competitively inhibits tnf signaling. overexpression of dcr in tumor cells protects them from apoptosis. dcr protects tumor cells from immune surveillance as it contributes to the suppression of the host anti-tumor immunity. dcr mrna and protein are amplified in various malignant tissues, such as lung cancer, colon cancer, gastric cancer, oesophageal carcinoma, pancreas cancer and malignant melanoma. wu et al. reported that dcr could not be detected in non-tumor patients, but could be detected in % of patients with malignant cancers. this phenomenon demonstrates that the elevated dcr expression is significantly correlated with tumorigenesis and tumor progression. wu et al. reported that dcr was highly expressed in human gastric cancer, and positively correlated with the development and metastases of gastric lesions. gastric cancer patients with a high dcr expression presented a more advanced pn2- disease than those with a low dcr expression. the dcr for fasl may be involved in the progression of gastric cancer. further evaluation of the possible roles of dcr and the regulation of dcr expression in malignant cells is very important for the development of new strategies for controlling the growth of malignant cells that escape the host immune surveillance. at the early stage, the mapk/erk pathway is activated in tumors, which is a notable sign for many kinds of cancers in humans. recently, several lines of evidence suggested that erk could be a parameter for predicting the prognosis of various cancers such as breast cancer, colon cancer, pancreas cancer and cholangiocarcinoma. wang et al. reported that the expression of erk1/ was high in cholangiocarcinoma, which correlated with the tnm stages. kim et al. found that lps induced dcr release in human intestinal epithelial cells, which appeared to be via the activation of mitogen-activated protein kinases, such as extracellular signal-regulated kinase and and c-jun nh2-terminal protein kinase. lps-induced dcr release in sw cells was abolished by erk1/ and jnk inhibitors. furthermore, yang et al. reported that 3 g/ml dcr markedly induced the phosphorylation of erk and p in accordance with these reports, we propose that dcr and erk1/ are closely related. the dcr genes closely correlated with the occurrence and development of many kinds of tumors. in this study we investigated the expression level and location of dcr and erk1/ in gastric cancer patients of different ages, gender, stage and differentiation, to explore the relationship between dcr3/erk1/ and gastric cancer occurrence and development. furthermore, we provide suggestions for clinical diagnosis of gastric cancers. methods clinical samples tumors were from gastric cancer patients who were undergone endoscopic biopsy or curative operations in zhongshan hospital affiliated with xiamen university. tumor tissues from which dna and protein were isolated were from fresh specimen of resection surgery. all samples were obtained with patient consent and approval of the committee on medical ethics of zhongshan hospital xiamen university. mice all the experiments were performed using 6–8 weeks male nude mice purchased from model animal research center of medical college xiamen university. all the animals were housed under specific pathogen free conditions with constant access to water and chow. all experimental procedures were carried out following the approval of the animal care and use committee of xiamen university. cell culture the human gastric cancer cell line bgc was maintained in our laboratory, which was cultured in flasks with dulbecco’s modified eagle’s medium supplemented with 10% fetal bovine serum, and 1% penicillin-streptomycin at 37°c in an humidified atmosphere of 5% co reagents dmem, fbs and penicillin-streptomycin were purchased from hyclone corporation. hematoxylin-and-eosin assay kit were purchased from chemicon international, inc. erk1/ and dcr abs were purchased from santa cruz bio technology. rt-rcr reagents were purchased from takara. the primer sequence was synthesized from sangon. in vivo animal tumor models tumors were generated in male nude mice by intramuscular injection of bgc cells into the right flank. tumor measurements were converted to tumor volume by the formula, where l and w are the length and width, respectively. measurements were made with a vernier caliper. all tumor-bearing mice were divided randomly into groups. rt-pcr and western blotting analysis for examining the expression of dcr3/erk total rna of dcr and erk1/ was extracted from stimulated cells using trizol. to measure erk/dcr gene copy number, dna from fresh tumor samples was analyzed with rt-pcr. the upstream and downstream primers for erk mrna were 5'-cctgctcatcaacaccacc-3’ and 5'-cgtagccacatactccgtca-3’, and for erk mrna were 5'-tcttcc agccctccttcctg-3' and 5'-cgtttctgcgccgttaggt-3'. the samples were denatured at 95°c for 4 min, followed by cycles of amplification. the products were 300-bp and 400-bp fragments respectively. for dcr mrna, the upstream primer was 5'- gcaaagccaaggattccccctg -3’ and the downstream primer was 5'-ggcactgctctgagctggagctg-3’. the samples were denatured at 95°c for 4 min, followed by cycles of amplification. the product was a 921-bp fragment. bgc cells were harvested and lysed in cell lysis buffer nonidet p- 150 mm nacl, 50 mm tris–hcl, 1 mm pmsf, 2 g/ml aprotinin, and 2 g/ml leupeptin) which could release dcr and erk1/ proteins. twenty-five micrograms of total lysate was fractionated by sds-page and subjected to western blotting analysis using the anti-erk or anti-dcr mab. western blotting and elisa assay for examining the expression of dcr3/erk after inhibitors treatment bgc cells culture supernatants were collected at various intervals, and levels of u pd apdc, mek1/ and erk1/ interferences were quantified using commercial elisa kits, according to the vendor’s instructions. cells treated with erk1/ shrna, u0126/pd and apdc, respectively. after a 5- or 7-day incubation, cells were subjected to the cytokines as indicated assay. twenty-five micrograms of total lysate was fractionated by sds-page and subjected to western blotting analysis using the anti-erk or anti-dcr mab. immunohistochemistry analysis for the expression of dcr and erk1/2 tumor tissues were fixed in a formaldehyde medium and embedded in paraffin. sections mm thick were mounted on glass slides pretreated with % poly-l-lysine. they were then deparaffinaged in xy-lene, dehydrated in graded ethanol and soaked in 3% h2o for 10 min to eliminate endogenous peroxidase activity. next, the slides were submerged in citrate buffer and boiled at 92–98°c in a microwave oven for 10 min. subsequently, they were rinsed times with pbs for 10 min each and blocked with 10% normal goat serum in pbs for 1 hr at room temperature. the slides were then reacted with the affinity-purified rabbit anti-tr ab at room temperature for 2 hr. after washing, the slides were incubated with biotinylated goat-anti-rabbit antibody for 10 min. tr signal was revealed by streptavidin-peroxidase using dab as a substrate according to instructions from the histostain-plus kit. dcr signals were revealed in brown. finally, the slides were counterstained with hematoxylin and sealed with aqueous mounting media. statistical analysis data were presented as mean ± sd. the significance of the difference between the groups was assessed by student`s two-tailed t-test. probability value of less than was considered significant. all means were calculated from at least three independent experiments.  cancer patients have elevated dcr levels to study the correlation between dcr expression and tumor occurrence and development, tumors from gastric cancer patients were collected and tested for dcr mrna and protein levels. as shown in figure 1a, the 921 bp dcr bands were generated by rt-pcr from the tumor tissues of most patients, compared with the non-cancerous tissues from the same organs of these patients. these results demonstrate that dcr levels were significantly increased. to further confirm our results, the dcr protein levels were examined by western blotting. the results demonstrate that dcr protein could be detected in most cancer patients; dcr protein was detected in 74% of the tumor tissues, but only in 6% of the non-cancerous tissues. expression of erk1/ in clinical samples to identify the signaling molecules related to dcr the expression levels of erk1/ jnk and p were tested. we found that only erk1/ could be detected in tumor samples. erk mrna was detected in 74% of the samples, and erk mrna was detected in 64% of the samples. at the protein level, erk was detected in 84% and erk in 74% of the tumor tissues. these results indicate that patients with gastric cancer tumors have a higher erk1/2-positive occurrence compared with the non-cancerous tissues. there was a positive correlation between the mrna and protein levels, as in of tumor tissues the erk protein level was elevated, of which cases were consistent with the mrna expression. in contrast, erk protein expression was detected only in five normal tissues. when examining erk of cases of tumor tissues were positive for erk protein expression, of which cases were consistent with the erk mrna expression, while only ten cases showed positive protein expression in normal tissues. the eight randomly picked samples are shown in figure location of erk1/ in gastric cancer patients to examine erk1/ distribution, tumors from patients were tested by immunohistochemistry. the results show that erk1/ were expressed in tumor tissues from most of the patients. erk expression was found in the cytoplasm. erk positive expression was found in some tumor cells. dcr and erk1/ levels correlate with tumor invasion but not with age, gender or differentiation the positive incidence of dcr mrna in tumor tissues of gastric cancer patients was %, and of dcr protein %, which was significantly higher than in non-cancerous tissues showing only 4% and 6%, respectively. the positive occurrence of erk1/ mrna expression was % and %, and of protein expression % and %. both of them were significantly higher than the non-cancerous tissues, showing % and %, and % and %, respectively. these results suggest that dcr and erk1/ expression levels correlated with tumor occurrence and development. as shown in tables and there was no significant difference between tumor tissues from different ages, gender groups and patients with different differentiation stages of gastric cancer. however, dcr and erk1/ expression levels were significantly high in tnm stage ii-iv. thus, the expressions of dcr and erk1/ correlated with tumor invasion and tnm stage, but not with age, gender or differentiation. expression levels of dcr and erk1/ are amplified in animal models as shown in table after injecting bgc cells into the right flank of nude mice, the tumors grew each day. rt-pcr was used to test dcr and erk1/ mrna levels in the tumors and western blot analysis was used to examine protein levels. in gastric cancer animal models, dcr erk and erk were detected at 921 bp, 300 bp and 400 bp, respectively. in these tumor tissues dcr mrna was detected from day six, peaked on day and remained detected on day dcr was also detected from day six to day in liver, while it was only detected on day in heart and lung. erk1/ mrna expressions were detected from day four in tumor tissues, and erk mrna peaked on day. note: △p <  vs normal; **p <  vs model. dcr protein was detected on day six in tumor tissues and liver, and the expression remained until day dcr protein was detected on day in spleen and by day in heart, kidney and lung. erk protein was detected on day four in tumor tissues, and continued to increase each day. erk protein remained at a stable level in heart, liver and kidney, but it decreased in lung and spleen on day after reaching the peak. erk was detected on day two in spleen and tumor tissues. from day four, erk protein was detected in all six samples, and continued to increase till day these results suggest that erk and erk might have different effects on tumor occurrence, development and clonal expansion. dcr expression decreased after inhibiting the expression or phosphorylation of erk1/ in bgc cells to investigate the effect of erk1/ expression and phosphorylation on dcr expression, bgc cells were treated with erk1/ shrna or with inhibitors that specifically regulate the erk pathway. western blot analysis confirmed that the inhibitors efficiently blocked the phosphorylation of the mek/erk pathway molecules, and that the shrna significantly reduced the expression of erk1/. as shown in figure 5a, when bgc cells were treated with erk1/ shrna, erk1/ and p-erk1/ levels declined compared with the control. u is a very effective mek inhibitor, resulting in the inhibition of erk phosphorylation, as does pd erk phosphorylation gradually declined as the concentrations of the drugs increased, although total erk1/ protein expression hardly changed. apdc can inhibit nf-κb cell activation in a variety of cells. through the degradation of iκb, apdc can decrease the translocation of nf-κb, thus blocking nf-κb activation. as shown in figure c, under different concentrations of apdc, changing the level of nf-κb inhibition can significantly attenuate erk1/ phosphorylation levels. however, the specific mechanism requires further investigation. to examine the effect of these inhibitors and shrna on dcr expression we used elisa analysis, which demonstrated that secreted dcr in the supernatant decreased after the different treatments. statistical analysis showed that dcr secretion levels were significantly different between the experiment groups and control groups. as shown in figure interference with erk1/ in bgc cells led to decreased dcr protein expression compared with the control group. the trend matches the erk expression level in figure and proves that the two are positively correlated. furthermore, dcr and p-erk expression levels decreased when cells were treated with different concentrations of u pd and apdc. this data indicates that secretion of dcr positively correlated with p-erk1/ expression levels in bgc gastric cells. it is worth noting that in the u group, dcr secretion levels increased when the drug concentration reached 40 μmol/l; however, the specific mechanism requires further investigation. in the apdc group, dcr levels did not change significantly at concentrations higher than 20 μmol/l. discussion it has been demonstrated that the dcr gene is expressed at a low level in human embryo, lung, brain, liver, spleen, stomach, colon, lymph nodes and spinal cord, whereas it was expressed at a high level in cancers such as gastrointestinal cancer, hepatocellular carcinoma and pancreatic cancer. wu et al. reported that the expression of dcr in gastric cancer patients was significantly higher than normal. dcr expression in the well-differentiated gastric cancer was significantly lower than that of poorly differentiated specimens. the dcr expression level was significantly associated with lymph node metastasis and pathological stage, but did not correlate with tumor size, metastatic status, or histological types. when patients were followed-up for 63 months, dcr overexpression was found to be associated with a significantly shortened survival rate. many reports have shown that high expression levels of erk1/ closely correlated with breast, colorectal and pancreatic cancer, as well as malignant melanoma, leukemia and myxoma. our research showed that in patients with gastric cancer, the positive incidence of dcr and erk1/ mrna was higher than that in the non-cancerous tissues. rt-pcr and western blotting showed that the mrna and protein expression levels of dcr and erk1/ in tumor tissues were significantly higher than those in non-cancer tissues, suggesting that dcr and erk1/ levels correlate with tumor development but not with age, gender or differentiation. our results showed that the positive incidence of expression of dcr and erk1/ mrna and dcr and erk1/ protein matched each other. the immunohistochemistry results also demonstrated that in gastric tumors, erk1/ was highly expressed. chen et al. reported that the positive rate of dcr expression was % in hepatocellular carcinoma, and that there was a significant correlation between dcr expression and metastasis as well as recurrence and differentiation. in the mouse gastric model, rt-pcr showed that dcr mrna could be detected in tumors from day six. erk1/ mrna and protein were also detected in tumors, and erk levels gradually increased in gastric tumors. furthermore, they were also detected in heart, liver, spleen, lung and kidney of the gastric cancer animal model, suggesting that they have an important role in tumor progression. erk1/ mrna was detected from day four in tumor tissues, and erk mrna peaked on day erk protein could also be detected on day four in tumor tissues, and continued to increase every day. erk remained at a stable level in heart, liver and kidney, but it decreased in lung and spleen on day after reaching the peak. erk was detected on day two in spleen and tumor tissues. from day four, erk was detected in all six tissues, and continued to increase till day erk could not be detected in heart, lung and spleen by rt-pcr on day in animal models, but protein levels could be detected. these results suggest that erk and erk might have different effects on tumor occurrence, development and clonal expansion. many studies indicated that the expression of erk1/ mrna and protein varies in different tumors and cells. some recent reports suggested they could be entirely opposite in some cases. thus, the effects of erk and erk on the tumors are unlikely to be the same.  in conclusion, high expression of dcr and erk1/ may suppress tumor cell apoptosis and play an influential role in gastric cancer occurrence and development, which is an important mechanism in tumorigenesis. in our study, dcr and erk1/ presented an overexpression tendency, and participated in the tumor immunity. we infer that in the tumor occurrence and developing process, the expression of erk1/ and dcr may be related to each other. understanding the role of erk1/ in dcr expression might shed light on gastric cancer's diagnosis and identify a factor that regulates the expression of dcr it might be a new marker for early diagnosis of gastric cancer. dcr is a secreted protein, which can be detected in blood serum. thus it could serve as a reliable index for clinical malignant tumor diagnosis, treatment and prognosis. therefore, dcr has the potential of becoming a novel tumor marker in the future. competing interest the authors declare that they have no competing interest. authors’ contributions dy and xf carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. py, qw carried out the immunoassays. fy, sy and bl participated in the sequence alignment. zl participated in the design of the study and performed the statistical analysis. gz conceived of the study, and participated in its design and coordination. all authors read and approved the final manuscript. 
decoy receptor, a member of the tumor necrosis factor receptor superfamily, is associated with anti-tumor immunity suppression. the mapk/mek/erk pathway has been reported to be a regulator in tumor occurrence, development and clonal expansion. the expression of dcr and erk1/ in tumor tissues of gastric cancer patients was significantly higher than the non-cancerous group. there was no statistical difference among tumor tissues from patients with different ages or gender, and even of different differentiation. however, in patients with stage i gastric cancer, the dcr and erk1/ levels were significantly lower than patients with more advanced stages. dcr and erk1/ play a vital role in the development of gastric cancer, and they may be new markers for indicating the efficiency of gastric cancer treatment in the future.
 decoy receptor, a member of the tumor necrosis factor receptor superfamily, is associated with anti-tumor immunity suppression. it is highly expressed in many tumors, and its expression can be regulated by the mapk/mek/erk signaling pathway. the mapk/mek/erk pathway has been reported to be a regulator in tumor occurrence, development and clonal expansion. external-signal regulated kinase is a vital member of this pathway.  the expression of dcr and erk1/ in tumor tissues of gastric cancer patients was significantly higher than the non-cancerous group. there was no statistical difference among tumor tissues from patients with different ages or gender, and even of different differentiation. however, in patients with stage i gastric cancer, the dcr and erk1/ levels were significantly lower than patients with more advanced stages.  dcr and erk1/ play a vital role in the development of gastric cancer, and they may be new markers for indicating the efficiency of gastric cancer treatment in the future. 