| Background:Gastric cancer(GC)is one of the most common malignancies worldwide,and GC cases in China account for almost 50%of all global cases.Because there are few symptoms in its early stages,most patients are diagnosed at advanced stages.Five-year survival is less than 20%.Poor prognosis for GC is due to significant invasiveness and metastases.Metastasis is the major cause of carcinoma-induced death,but mechanisms are poorly understood.Epithelial-to mesenchymal transition(EMT)is a process necessary for morphogenesis during embryonic development and tumor metastasis in epithelial cancers.The process is characterized by leading polarized,immotile epithelial cells that convert to motile and invasive mesenchymal cells.During EMT,epithelial cells lose their marker E-cadherin and gain mesenchymal markers such as Vimentin.Transforming growth factor-?(TGF-?)is an extracellular cytokine that known to initiate EMT in various cancers to promote tumor metastasis.Homeobox genes are a large and essential family in the developmental regulatory hierarchy.Data show that they are aberrantly expressed in various solid tumors.Dysregulated expression of these embryonic transcription factors has been thought to be associated with tumor development and progression.Bapx1,a homeobox-containing gene located on chromosome 4p15.33,is a NKX family member most closely related to Drosophila bagpipe and is essential for morphogenesis of the gastroduodenal tract.In Bapx1-/-mouse embryos,stomach epithelia lacks antral gland units.In addition,Bapxl overexpression is significantly associated with distant metastasis and chemoresistance in ovarian carcinoma,but its function in GC is unknown.Bapx1 is thought to act downstream of BMP and TGF-?in embryonic cells.Therefore,we hypothesized that a functional interaction between Bapx1 and TGF-? may affect EMT in GC.The purpose of this study was to investigate the effects and mechanism of Bapxl on EMT in human GC.Methods:The expression of Bapxl in several GC cell lines,fresh samples,stored specimens of human GC and matched adjacent noncancerous gastric tissues was assessed by Western blot and immunohistochemistry.In vitro,Bapx1 was silenced in BGC823 and MGC803 cells.The effect of Bapxl on migration,invasion and EMT phynotypes(E-cadherin,Vimentin,MMP9 and ?-catenin)in gastric cancer cells were evaluated by Transwell,Wound closure assays and Western Blot.In the presence of TGF-?,the cell invasion and/or migration were assessed.Then,Bapxl and EMT-related proteins were analyzed by Western blot.To verify the role of Bapx1 in TGF-?induced EMT,we silenced Bapx1 in BGC823 and MGC803 cells and then treated with TGF-?1 for 72 h and the control group we treated with siRNA.And the normal group cells we treated with neither siBapx1 nor TGF-?.We evaluated the migration and invasion ability in all the 3 groups in both BGC823 and MGC803 by transwell.The morphological changes of the cells were also observed.Next,Western blot was used to identify differences in EMT response kinetics after Bapxl depletion.The activity of NF-?B was evaluated by Immunofluorescence staining and Western blotting of nucleoprotein.Experiments were repeated at least 3 times.Data are presented as the mean ± SEM.The significance of differences was analyzed by using one-way analysis of variance or Student's t-test.Correlation between the differential classification and Bapxl score were examined by Spearman correlation coefficients.Kaplan-Meier method was performed to evaluate the survival curve,and log-rank test was used to estimate the differences between survival curves.Statistical analyses were performed with SPSS software.For all comparisons,2-sided P<0.05 was considered statistically significant.Results:Bapx1 expression was upregulated in MKN45,MGC803,BGC803,BGC823 and SGC7901 GC cells compared to immortalized human gastric epithelial line GES1.Bapxl expression was greater in GC tissues compared to adjacent non-tumor tissues.High Bapxl expression was more frequent in patients with more advanced N stage(P = 0.007),M stage(P<0.001),and overall TNM stage(P =0.03).However,no statistical significance was found in T stage(P = 0.134).Average Bapx1 expression scores were also significantly higher with more advanced GC of N,M and overall TNM stages but not T stage.Indicated that Bapx1 may be associated with tumorigenesis of GC and may contribute to tumor metastasis.Kaplan-Meier analysis revealed that patients with strong Bapx-1 staining had significantly poorer overall survival(OS)compared with patients with negative Bapx1 expression(P<0.05).Subgroups of IV stage patients with positive Bapx1 expression had worse overall survival compared with those with negative Bapx1 expression.Silencing Bapx1 in BGC823 and MGC803 diminished cell invasion/migration and decreased mesenchymal phenotypes,which means significant upregulation of E-cadherin expression and concomitant downregulation of vimentin,?-catenin,and MMP9 expression in gastric cells.Transforming growth factor-?(TGF-(3)increased Bapx1 expression and epithelial-mesenchymal transition(EMT)in GC cells.TGF?1 also changed cell morphology from a more epithelial-like appearance to a mesenchymal-like spindle-cell shape and increased intercellular separation.However,down-regulated Bapxl reversed TGF-p induced EMT,as shown in migration/invasion assays and morphological changes.siBapx1 substantially abolished TGF-?1-stimulated upregulation of vimentin,?-catenin,MMP9 and down regulation of E-cadherin,indicating that Bapxl participates in EMT induced by TGF-?1.The western blot of nucleoprotein and immunofluorescence staining indicated that P65 is localized both in the nucleus and the cytoplasms in BGC823 and MGC803 cells.While siBapxl reduced the nuclear translocation of NF-?B.In summary,Bapx1 indicates poor prognosis for GC by promoting tumor migration and invasion via TGF-?-induced EMT.Conclusion:Bapx1 expression is significantly upregulated in GC and is correlated with TNM stages,metastasis,and poor OS.Silencing Bapx1 hinders GC cell invasion and migration and impairs EMT in vitro.Bapxl promotes TGF-p 1 induced EMT,and this affects the development of GC.Therefore,our findings suggest that Bapxl is an oncogene in GC,contributing to metastasis and poor survival among GC patients by facilitating TGF-?-induced EMT.Bapx1 targeting may be a potential predictive marker for GC patient outcomes and a potential therapeutic strategy to prevent GC metastasis. |
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