BACKGROUND
gastric carcinoma is the second most common causes of cancer-related deaths in the world, especially in asia  <cit> .
most of gastric cancer patients died of tumor recurrence caused by distant metastasis.
metastasis is a multi-stage process by which cancer cells disseminate from the primary neoplasm and invade surrounding tissue and distance organs, involving cancer cell motility, intravasation, transit in the blood or lymph and extravasation  <cit> .
this process depends tightly on the surrounding microenvironment and tumor development relies on a continuous cross-talk between cancer cells and extracellular microenvironments  <cit> .
transgelin  is an actin cross-linking protein that is involved in calcium interactions and regulates contractile properties  <cit> .
it may play a role in cell differentiation, cell migration cell invasion and matrix remodeling by stabilizing the cytoskeleton through actin binding  <cit> .
overexpression of tagln protein has been observed in carcinomas of the stomach, liver, and esophagus, while decreased levels of tagln mrna have been observed in breast and colon cancer cell lines and primary tumors  <cit> .
it is generally accepted that tumor-stroma interactions play an important role in tumor development and progression.
the stroma is constituted mainly of extracellular matrix  and cellular elements, such as fibroblasts  <cit> .
the cellular components of the stroma are well-recognized as having a supportive role in carcinogenesis  <cit> .
these stromal elements act in a synergistic cross-talk with cancer cells from the primary sites to sustain cancer growth and metastasis  <cit> .
the tumor stroma which is referred to as a “reactive stroma” is associated with an increased number of fibroblasts, enhanced capillary density and deposition of a new ecm rich in type-1-collagen and fibrin  <cit> .
accumulating evidence reveals that stromal cells, such as fibroblasts have a more profound influence on the development and progression of carcinoma than was previously appreciated  <cit> .
fibroblasts are well-known to play roles in tumorigenesis and progression.
fibroblasts express vimentin, fibronectin, α-smooth-muscle actin , fibroblast surface protein as well as fibroblastic markers.
carcinoma-associated fibroblasts  are activated fibroblasts, biologically different from fibroblasts present in benign microenvironments in several important aspects  <cit> .
cafs are not passive bystanders in tumorigenesis and metastasis, but contribute actively to these processes  <cit> .
our knowledge on the role of resting and activated fibroblasts in cancer is still evolving.
here, we focus on the interaction between cancer cells and their microenvironments to study how the fibroblasts isolated from human gastric carcinomas facilitate tumorigenesis.
RESULTS
the expression of tagln is upregulated in gastric carcinoma-associated fibroblasts
the carcinoma-associated fibroblasts , counterpart fibroblasts  and normal fibroblasts  were obtained from primary tissue culture using tissues from cancer-associated regions, non-cancer-associated stroma and normal mucosa, respectively.
we then verified the purity of the various fibroblast populations by immunostaining.
these fibroblast populations expressed high levls of fibroblastic markers such as vimentin, α-sma and fibronectin.
on the other hand, these cells did not express epithelial markers such as cytokeratin .
these data confirmed the purity of the fiborblasts.
these observations indicate that the culture cells were predominantly fibroblasts were prepared with minimal contamination by other cells.
the mrna and protein levels of tagln were lower in cpfs and nfs compared with cafs .
we first showed that cafs grew significantly more than cpfs and nfs in vitro cell proliferation .
next, we carried out cell invasion and migration assay to test whether the invasion and migration ability of gastric cancer cells would be enhanced by fibroblasts.
the experiment was designed to investigate the effects of stroma tagln, which was secreted by fibroblasts, on human gastric cancer cells.
the internal levels of tagln in gastric cancer cells might interfere the experimental process.
hence, we choosed mkn- <dig> cell line, because its tagln expression level was the lowest in seven gastric cancer cell lines .
interestingly, the invasion and migration ability of mkn- <dig> increased in all the three groups , and increased most in the group with caf .
the acquisition of migration and invasive behavior is one of steps in the metastatic process.
cafs promote tumor metastasis through upregulating tagln
to examine the effect of tagln in fibrobalsts, we used rnai to knock down tagln expression.
cafs, cpfs and nfs were transfected with tagln-specific or non-silence sirna.
rna and protein were obtained at  <dig>   <dig> or 72 hrs after transfection, tagln mrna and protein levels were investigated by qrt-pcr and western blot.
tagln mrna and protein were significantly inhibited in cells treated with tagln-specific sirna .
we next investigated whether the invasion and migration ability of gastric cancer cells will be enhanced by the expression of tagln in fibroblasts, we carried out cell invasion and migration assay in vitro.
the fibroblasts and mkn- <dig> were non-contactedly co-cultured.
interestingly, the invasion and migration ability of mkn- <dig> decreased immediately in the group with cafs and decreased slightly in the group with cpfs and nfs.
the results showed that the invasion and migration ability of mkn- <dig> can decrease through down-regulating the expression of tagln .
in order to assess the contribution of cafs-tagln to tumor metastasis in vivo, we emplyed a xenograft model.
we mixed tagln sirna cafs, non-silenced cafs, cpfs and nfs with mkn- <dig> human gastric cancer cells in a 2: <dig> ratio or mkn- <dig> alone and inoculated these cells through tail intravenous injection in immunodeficient nude mice.
after 6 weeks, mkn- <dig> mixed with cafs generated more lung metastatic nodules than mkn- <dig> mixed with tagln sirna cafs, cpfs and nfs, similar to mkn- <dig> mixed with non-silence sirna cafs .
these data suggested that cafs show an increased ability to stimulate tumor metastasis .
all the observations indicated that in the presence of cafs, tumors became more aggressive, suggesting that cafs might play roles in promoting metastasis in gastric cancer.
tagln promotes tumor metastasis by upregulating mmp-2
many studies have focused on the role of matrix metalloproteinases  to the invasion of surrounding connective tissue and metastasis of tumor cells from the primary lesion to distant sites in tumor progression  <cit> .
in our previous work, we examined mmp- <dig>  mmp- <dig>  mmp- <dig>  mmp- <dig> and mmp- <dig> levels after tagln knock-down by sirna through elisa, only mmp- <dig> level was significantly down-regulated, other mmps levels showed no change.
hence, we focued on mmp- <dig> in this study.
as figure 4a showed, compared to mkn- <dig> co-coultured with caf/neg and caf/mock, mmp- <dig> levels in supernatant from mkn- <dig> co-cultured with caf/sitagln were significantly suppressed.
we next examined the activity of mmp- <dig> using gelatin zymography assay.
we found mmp- <dig> activity was dramatically inhibited in supernatant from mkn- <dig> co-cultured with caf/sitagln than with caf/neg and caf/mock .
thus, tagln may enhance tumor metastasis through the mmp- <dig> enzymes degrade the basement membranes .
tagln expression in human gastric cancer and its association with differentiation in gastric cancer patients
to evaluate the tagln expression in gastric cancer, tagln immunostaining in  <dig> primary gastric cancer tissues were examined.
tagln expression was upregulated in human gastric cancer stroma compared to normal gastric tissue .
the correlation of tagln expression with the clinicopathological features was shown in table  <dig>  tagln levels were higher in undifferentiated tumors  than that in differentiated ones  .
moreover, tagln levels was correlated with lymph node metastasis .
there were no significant differences in tumor tagln expression according to the other clinicopathological features such as gender, age, tumor size, lauren classification, t stage, distance metastasis and tnm stage .
also, we used qrt-pcr and western blot to evaluate the expression of tagln in gastric cancer tissues .
these results suggested that the tagln expression in gastric cancer tissues was much higher in gastric cancer than that in paired normal tissues.
discussion
accumulating evidence demonstrates that cancer initiation and progression involve interactions between both tumor and stromal cells.
tumor cells can actively recruit stromal cells, such as vascular cells, smooth muscle cells and fibroblasts, into the tumor, thus generating microenvironment to foster tumor growth  <cit> .
fibroblasts often constitute the majority of the stromal cells within a gastric carcinoma, yet the functional contributions of these cells to tumorigenesis and tumor metastasis remain poorly understood.
tagln is one of the earliest markers of smooth muscle differentiation during embryogenesis  <cit> , and previous studies related to tagln mainly focused on smooth muscle cells  <cit> .
pathological conditions accompanied with tissue remodeling and fibrogenesis, such as wound healing and neoplastic lesions, are characterized by the appearance of stromal cells with ultrastructural features intermediate between those of typical fibroblasts and those of smooth muscle cells.
these fibroblasts exhibit phenotypic and functional features of smooth muscle cells and hence are named myofibroblasts  <cit> , such as cafs.
in our study, tagln overexpression was also observed in cafs.
it is reported that the tagln protein is overexpressed in gastric cancer when protein expression spectrum of gastric cancer was analyzed using two-dimensional gel electrophoresis   <cit> .
furthermore, the tagln protein is one of the two tumor-associated antigens which were identified in serum of kidney carcinoma patients by klade using 2-de  <cit> .
these reports are consistent with the findings that we have observed using real-time pcr and western blot.
interestingly, we found that tagln was mainly detected in fibroblasts derived from tumor stroma, rather than in tumor cells.
similarly, other investigators discovered that the tagln is not expressed in the malignant cells but in mesenchymal cells of the tumor stroma  <cit> .
stromal fibroblasts regulate endothelial or epithelial cell behavior through direct and indirect cell-cel <dig> interactions.
activation of host stroma <dig> microenvironment is thought to be a critical step in tumor growth and progression  <cit> .
proteins secreted by stromal cell in the tumors may positively or negatively affect tumor progression.
the most prominent actions of tagln may favor tumor cell motility and invasion  <cit> .
we found that the tagln was significantly increased in lymph node metastasis group than lymph node negative group.
to c1arify the role of tagln in stromal fibroblasts during gastric cancer tumorigenesis and metastasis, we silenced the tagln expression in human cafs.
it was indicated that only the mkn- <dig> co-cultured with cafs expressing high level tagln exhibited much higher invasion and migration ability.
fibroblasts which tagln had been knocked down did not display this function.
taken together, we suggest that tagln might be responsible for the enhancement of gastric cancer cell metastasis via stromal cells such as cafs.
tagln expression is increased significantly in gastric cafs.
cafs with increased tagln expression levels may enhance tumor cell invasion and migration ability which promote an enhanced expression of mmp- <dig>  the stimulation of mmp expression in fibroblasts either after direct contact with tumor cells or after contact with tumor cell-derived soluble factors from tumors originating from epithelial cells has been shown in several studies  <cit> .
clarifying the underlying mechanisms may therefore attenuate the tumor recurrence and metastasis in gastric cancer patients.
CONCLUSIONS
in summary, our study demonstrates that cafs may promote gastric cancer cell migration and invasion via increased mmp- <dig> production caused by tagln upregulation.

 gastric carcinoma is the second most common causes of cancer-related deaths in the world, especially in asia. most of gastric cancer patients died of tumor recurrence caused by distant metastasis. metastasis is a multi-stage process by which cancer cells disseminate from the primary neoplasm and invade surrounding tissue and distance organs, involving cancer cell motility, intravasation, transit in the blood or lymph and extravasation. this process depends tightly on the surrounding microenvironment and tumor development relies on a continuous cross-talk between cancer cells and extracellular microenvironments. transgelin is an actin cross-linking protein that is involved in calcium interactions and regulates contractile properties. it may play a role in cell differentiation, cell migration cell invasion and matrix remodeling by stabilizing the cytoskeleton through actin binding. overexpression of tagln protein has been observed in carcinomas of the stomach, liver, and esophagus, while decreased levels of tagln mrna have been observed in breast and colon cancer cell lines and primary tumors. it is generally accepted that tumor-stroma interactions play an important role in tumor development and progression. the stroma is constituted mainly of extracellular matrix and cellular elements, such as fibroblasts. the cellular components of the stroma are well-recognized as having a supportive role in carcinogenesis. these stromal elements act in a synergistic cross-talk with cancer cells from the primary sites to sustain cancer growth and metastasis. the tumor stroma which is referred to as a “reactive stroma” is associated with an increased number of fibroblasts, enhanced capillary density and deposition of a new ecm rich in type-1-collagen and fibrin. accumulating evidence reveals that stromal cells, such as fibroblasts have a more profound influence on the development and progression of carcinoma than was previously appreciated. fibroblasts are well-known to play roles in tumorigenesis and progression. fibroblasts express vimentin, fibronectin, α-smooth-muscle actin, fibroblast surface protein as well as fibroblastic markers. carcinoma-associated fibroblasts are activated fibroblasts, biologically different from fibroblasts present in benign microenvironments in several important aspects. cafs are not passive bystanders in tumorigenesis and metastasis, but contribute actively to these processes. our knowledge on the role of resting and activated fibroblasts in cancer is still evolving. here, we focus on the interaction between cancer cells and their microenvironments to study how the fibroblasts isolated from human gastric carcinomas facilitate tumorigenesis.  the expression of tagln is upregulated in gastric carcinoma-associated fibroblasts the carcinoma-associated fibroblasts, counterpart fibroblasts and normal fibroblasts were obtained from primary tissue culture using tissues from cancer-associated regions, non-cancer-associated stroma and normal mucosa, respectively. we then verified the purity of the various fibroblast populations by immunostaining. these fibroblast populations expressed high levls of fibroblastic markers such as vimentin, α-sma and fibronectin. on the other hand, these cells did not express epithelial markers such as cytokeratin. these data confirmed the purity of the fiborblasts. these observations indicate that the culture cells were predominantly fibroblasts were prepared with minimal contamination by other cells. the mrna and protein levels of tagln were lower in cpfs and nfs compared with cafs. we first showed that cafs grew significantly more than cpfs and nfs in vitro cell proliferation. next, we carried out cell invasion and migration assay to test whether the invasion and migration ability of gastric cancer cells would be enhanced by fibroblasts. the experiment was designed to investigate the effects of stroma tagln, which was secreted by fibroblasts, on human gastric cancer cells. the internal levels of tagln in gastric cancer cells might interfere the experimental process. hence, we choosed mkn- cell line, because its tagln expression level was the lowest in seven gastric cancer cell lines. interestingly, the invasion and migration ability of mkn- increased in all the three groups, and increased most in the group with caf. the acquisition of migration and invasive behavior is one of steps in the metastatic process. cafs promote tumor metastasis through upregulating tagln to examine the effect of tagln in fibrobalsts, we used rnai to knock down tagln expression. cafs, cpfs and nfs were transfected with tagln-specific or non-silence sirna. rna and protein were obtained at or 72 hrs after transfection, tagln mrna and protein levels were investigated by qrt-pcr and western blot. tagln mrna and protein were significantly inhibited in cells treated with tagln-specific sirna. we next investigated whether the invasion and migration ability of gastric cancer cells will be enhanced by the expression of tagln in fibroblasts, we carried out cell invasion and migration assay in vitro. the fibroblasts and mkn- were non-contactedly co-cultured. interestingly, the invasion and migration ability of mkn- decreased immediately in the group with cafs and decreased slightly in the group with cpfs and nfs. the results showed that the invasion and migration ability of mkn- can decrease through down-regulating the expression of tagln. in order to assess the contribution of cafs-tagln to tumor metastasis in vivo, we emplyed a xenograft model. we mixed tagln sirna cafs, non-silenced cafs, cpfs and nfs with mkn- human gastric cancer cells in a 2: ratio or mkn- alone and inoculated these cells through tail intravenous injection in immunodeficient nude mice. after 6 weeks, mkn- mixed with cafs generated more lung metastatic nodules than mkn- mixed with tagln sirna cafs, cpfs and nfs, similar to mkn- mixed with non-silence sirna cafs. these data suggested that cafs show an increased ability to stimulate tumor metastasis. all the observations indicated that in the presence of cafs, tumors became more aggressive, suggesting that cafs might play roles in promoting metastasis in gastric cancer. tagln promotes tumor metastasis by upregulating mmp-2 many studies have focused on the role of matrix metalloproteinases to the invasion of surrounding connective tissue and metastasis of tumor cells from the primary lesion to distant sites in tumor progression. in our previous work, we examined mmp- mmp- mmp- mmp- and mmp- levels after tagln knock-down by sirna through elisa, only mmp- level was significantly down-regulated, other mmps levels showed no change. hence, we focued on mmp- in this study. as figure 4a showed, compared to mkn- co-coultured with caf/neg and caf/mock, mmp- levels in supernatant from mkn- co-cultured with caf/sitagln were significantly suppressed. we next examined the activity of mmp- using gelatin zymography assay. we found mmp- activity was dramatically inhibited in supernatant from mkn- co-cultured with caf/sitagln than with caf/neg and caf/mock. thus, tagln may enhance tumor metastasis through the mmp- enzymes degrade the basement membranes. tagln expression in human gastric cancer and its association with differentiation in gastric cancer patients to evaluate the tagln expression in gastric cancer, tagln immunostaining in primary gastric cancer tissues were examined. tagln expression was upregulated in human gastric cancer stroma compared to normal gastric tissue. the correlation of tagln expression with the clinicopathological features was shown in table  tagln levels were higher in undifferentiated tumors than that in differentiated ones. moreover, tagln levels was correlated with lymph node metastasis. there were no significant differences in tumor tagln expression according to the other clinicopathological features such as gender, age, tumor size, lauren classification, t stage, distance metastasis and tnm stage. also, we used qrt-pcr and western blot to evaluate the expression of tagln in gastric cancer tissues. these results suggested that the tagln expression in gastric cancer tissues was much higher in gastric cancer than that in paired normal tissues. discussion accumulating evidence demonstrates that cancer initiation and progression involve interactions between both tumor and stromal cells. tumor cells can actively recruit stromal cells, such as vascular cells, smooth muscle cells and fibroblasts, into the tumor, thus generating microenvironment to foster tumor growth. fibroblasts often constitute the majority of the stromal cells within a gastric carcinoma, yet the functional contributions of these cells to tumorigenesis and tumor metastasis remain poorly understood. tagln is one of the earliest markers of smooth muscle differentiation during embryogenesis, and previous studies related to tagln mainly focused on smooth muscle cells. pathological conditions accompanied with tissue remodeling and fibrogenesis, such as wound healing and neoplastic lesions, are characterized by the appearance of stromal cells with ultrastructural features intermediate between those of typical fibroblasts and those of smooth muscle cells. these fibroblasts exhibit phenotypic and functional features of smooth muscle cells and hence are named myofibroblasts, such as cafs. in our study, tagln overexpression was also observed in cafs. it is reported that the tagln protein is overexpressed in gastric cancer when protein expression spectrum of gastric cancer was analyzed using two-dimensional gel electrophoresis. furthermore, the tagln protein is one of the two tumor-associated antigens which were identified in serum of kidney carcinoma patients by klade using 2-de. these reports are consistent with the findings that we have observed using real-time pcr and western blot. interestingly, we found that tagln was mainly detected in fibroblasts derived from tumor stroma, rather than in tumor cells. similarly, other investigators discovered that the tagln is not expressed in the malignant cells but in mesenchymal cells of the tumor stroma. stromal fibroblasts regulate endothelial or epithelial cell behavior through direct and indirect cell-cel interactions. activation of host stroma microenvironment is thought to be a critical step in tumor growth and progression. proteins secreted by stromal cell in the tumors may positively or negatively affect tumor progression. the most prominent actions of tagln may favor tumor cell motility and invasion. we found that the tagln was significantly increased in lymph node metastasis group than lymph node negative group. to c1arify the role of tagln in stromal fibroblasts during gastric cancer tumorigenesis and metastasis, we silenced the tagln expression in human cafs. it was indicated that only the mkn- co-cultured with cafs expressing high level tagln exhibited much higher invasion and migration ability. fibroblasts which tagln had been knocked down did not display this function. taken together, we suggest that tagln might be responsible for the enhancement of gastric cancer cell metastasis via stromal cells such as cafs. tagln expression is increased significantly in gastric cafs. cafs with increased tagln expression levels may enhance tumor cell invasion and migration ability which promote an enhanced expression of mmp- the stimulation of mmp expression in fibroblasts either after direct contact with tumor cells or after contact with tumor cell-derived soluble factors from tumors originating from epithelial cells has been shown in several studies. clarifying the underlying mechanisms may therefore attenuate the tumor recurrence and metastasis in gastric cancer patients.  in summary, our study demonstrates that cafs may promote gastric cancer cell migration and invasion via increased mmp- production caused by tagln upregulation. 
tagln is an actin-binding protein that plays an important role in tumorigenesis. fibroblasts play a critical role in tumorigenesis, tumor progression and metastasis. we investigated the interaction between cancer cells and the tumor microenvironment to determine how the fibroblasts from human gastric carcinoma facilitate tumorigenesis through tagln. qrt-pcr and western blot indicated that tagln expression was upregulated in gastric carcinoma-associated fibroblasts that promote gastric cancer cell migration and invasion. cafs may promote gastric cancer cell migration and invasion via upregulating tagln and tagln induced mmp- production. tagln levels were elevated in human gastric cancer stroma than normal gastric stroma and associated with differentiation and lymph node metastasis of gastric cancer. using small interfering rna, we found that cafs enhanced tumor metastasis through upregulated tagln in vitro and in vivo.
 fibroblasts play a critical role in tumorigenesis, tumor progression and metastasis. however, their detailed molecular characteristics and clinical significance are still elusive. tagln is an actin-binding protein that plays an important role in tumorigenesis.  we investigated the interaction between cancer cells and the tumor microenvironment to determine how the fibroblasts from human gastric carcinoma facilitate tumorigenesis through tagln. qrt-pcr and western blot indicated that tagln expression was upregulated in gastric carcinoma-associated fibroblasts that promote gastric cancer cell migration and invasion. using small interfering rna, we found that cafs enhanced tumor metastasis through upregulated tagln in vitro and in vivo. the expression of matrix metalloproteinase- was significantly lower after tagln knock-down by sirna. tagln levels were elevated in human gastric cancer stroma than normal gastric stroma and associated with differentiation and lymph node metastasis of gastric cancer.  cafs may promote gastric cancer cell migration and invasion via upregulating tagln and tagln induced mmp- production. taglnfibroblastmicroenvironmenttumor metastasisgastric carcinoma 