Tbx2 Over-expression Promotes Proliferation,Invasion And Epithelial-mesenchymal Transition In Human Gastric Cancer

Objective: We analyzed the relationship between distribution of Tbx2 status in gastric cancer according to clinicopathological characteristics and investigated the effects of biological behavior in gastric cancer cells by TBX2.Proved that TBX2 is an important oncogene in gastric cancer and provided a new target for the diagnosis and treatment of gastric cancer.Methods: We selected 161 cases of primary gastric cancer samples and 10 cases of normal gastric tissues from patients undergo surgeries were implemented in the First Affiliated Hospital of China Medical University who received no preoperative radiotherapies and chemotherapies from 2008-2010.Tissues were fixed in 10% neutral buffered formalin,paraffin embedded.We analyed the relationship between gastric cancer expression of TBX2 with clinical and pathological factors in gastric cancer by immunohistochemistry.Gastric cancer cell lines included SGC-7901,BGC-823,AGS,GES1,MKN-1 were bought from ATCC and the Chinese Academy of Sciences Committee on Type Culture Collection cell bank.We observed the expression of TBX2 in gastric cancer cells by cell transfection techniques,real-time quantitative PCR and Western blot.We observed the proliferation situation of gastric cancer cells by clonogenic experiments and MTT assay after cell transfection.We observed the invasion situation of gastric cancer cells by matrigel transwell experiments after cell transfection.Result: TBX2 over-expressed in gastric cancer tissue: we detected the expression of TBX2 protein in 161 cases of gastric cancer patients and 10 cases of normal gastric tissues by immunohistochemistry.The results showed that TBX2 protein were present staining in both cell nucleus and cytoplasm.In 161 cases of gastric cancer patients,55.9%(90/161 patients)expression of TBX2 showed positive;while in normal gastric tissues the expression of TBX2 showed negative.The relationship between expression of TBX2 protein and clinical and pathological factors of gastric cancer: further analyzed the correlation between TBX2 expression and clinical and pathological factors,including age of patients,gender of patient,histological grade,TNM staging.We found that over-expression of TBX2 was related with higherTNM stage and tumor size and lymph node metastasis,but we found no significant correlation with other factors.Logistic analysis showed expression of TBX2,clinical stage and recurrence can significantly affected prognosis of patients.Cox multivariate regression analysis showed that clinical stage and recurrence could independently predicted prognosis of patients.Survival curves and Log-rank test showed expression of TBX2 was significantly associated with poor prognosis of patients.TBX2 promotes proliferation and invasion of gastric cancer cell : We detected endogenously expression of TBX2 in gastric cancer cell lines by western blot,it showed that expression amount was higher in BGC-823 cell,while the endogenous expression was lower in SGC-7901 cell lines.We treated these two cells separately by TBX2 plasmid transfection and si RNA interference,results showed that after TBX2 plasmid could be significantly increased m RNA and the corresponding protein expression of TBX2,while interfered with si RNA could significantly reduced m RNA and protein expression of TBX2.By MTT assay,we found that after TBX2 transfection could enhanced the proliferation ability of SGC-7901 gastric cancer cell,and TBX2 specific si RNA interference could significantly inhibited the proliferation ability of BGC-823 cells.In order to explored the role for cell invasion TBX2 played,we performed transwell experiments,the results showed that the invasive ability of cells after TBX2 plasmid transfection significantly enhanced,while invasion capacity of the cells reduced after interference of TBX2.TBX2 regulates invasion-related proteins: By vitro experiments we up-regulated and inhibited the expression of TBX2,and then screening expression changed associated with proliferation and invasion proteins.Results showed that compared with the control cells,TBX2 over-expression could significantly increased the expression of MMP2 and MMP9 protein and interferenced of TBX2 inhibited the expression of these proteins.The results explained that TBX2 could regulated proliferation and invasion of gastric cancer cells by MMP2 and MMP9 proteins.TBX2 regulates epithelial-mesenchymal transition: We detected expression of factors related with epithelial-mesenchymal transition,the results showed that TBX2over-expression down-regulated E-cadherin levels of SGC-7901,and interference of TBX2 slightly increased the expression of E-cadherin.Further confirmed that in gastric cancer cell lines,TBX2 could promote malignant phenotype occurred through ERK-MMP-2 signaling pathway,and could speed up intercellular epithelial to mesenchymal transition process.TBX2 regulates ERK signaling pathway: We studied MAPK signaling pathways: JNK,p38 and ERK,these signaling pathways regulated the expression of MMP-2.We found TBX2 over-expression could significantly increased the level of ERK phosphorylation,indicating that the expression of MMP-2 up-regulated by TBX2 induced activation of ERK signaling pathway.TBX2 regulates cell cycle-related proteins: We studied the change situation of cell cycle related protein.We found TBX2 over-expression could up-regulated the expression of cyclin E levels,and the expression of cyclin E was down-regulated while TBX2 was knockdown.The expression of p21 was down-regulated after TBX2 transfected,and up-regulated after TBX2 was knockdown.Indicated TBX2 promotes prolification and invasion likely through inhibition of p21 and up-regulation of cyclin E to achieved.Conclusion: In progression of gastric cancer,TBX2 can be over-expressed in human gastric cancer.TBX2 over-expression promotes the growth and invasion of gastric cancer cells.TBX2 mediates invasion,metastasis and EMT of gastric cancer cells via ERK-MMP.It can also mediate cell proliferation by down-regulate expression of p21 protein and up-regulate expression of cyclin E protein.