Effect Of Tiam 1 On The Invasion And Metastasis Of Gastric Cancer And Its Mechanism

Objective: Gastric cancer is one of the most common malignant tumors, its prognostic bad and survival rate low, the invasion and metastasis of which is one of the most important causes related to recurrence after surgery and high mortality. The invasion and metastasis of gastric cancer is a complex biological process with multiple phases and steps, in which locomotion and invasiveness are necessary for gastric cancer cells to metastasize. T lymphoma invasion and metastasis inducing factor 1(Tiam 1) is an important member of Dbl oncogene family, whose biological function is to regulate cytoskeletal reorgnization, body polarization, motion and migration, which is mediated by Rac 1,an important member of Rho oncogene family. Recent studys showed that proteins of Rho family were transducers and cellular switchs in vital movement, and they actively took part in the pathobiological process of tumor invasion and metastasis. As an upstream regulator of Rac 1, the possible roles of Tiam 1 in tumor invasion and metastasis became more and more outstanding and caught the attention of researchers. But up to now, these questions are still not clear. So in this study, we firstly detected the expression of Tiam 1 in gastric cancer tissues and analysed its relationship with the clinicopathological parameters. Then we examined the expression of Tiam 1 in gasric cancer cells with different invasive and metastatic potentials and analysed the correlations between them. Thirdly, we down-regulated the expression of Tiam 1 gene in gastric cancer cells and observed subsequent changes in their biological characters and the expressions of related factors. Through this research, We hope to explore the possible role of Tiam 1 in the invasion and metastasis of gstric cancer and its mechanism. Methods: (1) SABC immunohistochemistry were used to evaluate the expressions of Tiam 1 and Rac 1 in 60 gastric cancer tissues and normal paracarcinoma gastric mucosa(part from 3~5cm), meanwhile relationships among Tiam 1, Rac 1 and clinicopathological parameters were investigated. (2) Two subpopulations with different affinity to laminin(LN) were separated from human gastric cancer cell line MKN-45 by adhesion screening in vitro, the differences in cytoskeletal morphology, the composition of cell cycles and the invasive and metastatic potentials both in vitro and in vivo between them were observed. Then the expressions of Tiam 1 mRNA and protein were detected by RT-PCR, flowcytometry and cellular immunohistochemistry, at the same time correlations between the expression of Tiam 1 and the invasive and metastatic potentials of gastric cancer cells also were analysed. (3) A 18mer ASODN, trgeted to the specific sequence of Tiam 1 mRNA, was designed and transfected into gastric cancer cells with higher invasive and metastatic potentials using DOTAP liposomal transfection reagent. The expressions of Tiam 1 mRNA and protein were detected by RT-PCR and flowcytometry, meanwhile changes in cellular morphology, proliferation activity, cellular affinity and the invasive and metastatic potentials both in vivo and in vitro after transfection were observed too. Then the expressions of Rac 1, integrinβ1, 67kDa LNR, NF-κB and MMP-2 / -9 in transfected or no transfected gastric cancer cells with higher invasive and metastatic potentials and cells with lower invasive and metastatic potentials, were detected using flowcytometry and cellular immunohistochemistry technology. Results: (1) There was negative staining of Tiam 1 protein in paracarcinoma gastric mucosa, while positive staining was detected in gastric cancer tissues(P < 0.01). The positive staining rate of Rac 1 in gastric cancer tissues was significantly higher than that in paracarcinoma gastric mucosa(P < 0.01). As the degree of histologic differentiation of gastric cancer decreased, depth of invasion increased, stage of TNM upgraded or lymph node metastasis appeared, positive staining rates of Tiam 1 and Rac 1 proteins in gastric cancer tissues significantly increased(P < 0.05 or P < 0.01), meanwhile there were positive correlations between their expression(r = 0.567,P < 0.01). (2) Two subpopulations with different affinity to laminin(LN) could be separated from human gastric cancer cell line MKN-45 by adhesion screening in vitro. The one with lower affinity possessed a more regular cytoskeletal network, fewer actin bodys and pseudopodia, but the other one with higher affinity was superior in proliferation activity and the invasive and metastatic potentials both in vitro and in vivo(P < 0.05 or P < 0.01). The expression of Tiam 1 in cells with higher affinity was more intensive than that in cells with lower affinity(P < 0.05), meanwhile positive correlations existed between the expression of Tiam 1 and the invasive and metastatic potential of gastric cancer cells(r = 0.995~1,P < 0.01). (3) The expressions of Tiam 1 mRNA and protein were decreased markedly after Tiam 1 ASODN transfection using DOTAP liposomal transfection reagent( P < 0.01) and the inhibitive ratio was 79.8%and 71.3% respectively. After transfection,the cytoskeletal network of cells with higher invasive and metastatic potentials changed from disorder to regularity, and the actin bodys, cellular surface projections or pseudopodia diminished. Meantime, the cellular affinity to extracellular matrix and the invasive and metastatic potentials both in vitro and in vivo also decreased in some degree(P < 0.05 or P < 0.01). Examinations showed the expressions of Rac 1, integrinβ1, 67kDa LNR, NF-κB and MMP-2 / -9 in cells with higher invasive and metastatic potentials were more significantly intensive than that in cells with lower invasive and metastatic potentials(P < 0.05 or P < 0.01), but obvious differences only existed in the expressions of 67kDa LNR, NF-κB and MMP-2 / -9 in Tiam 1 ASODN transfected or no transfected cells with higher invasive and metastatic potentials(P < 0.05 or P < 0.01). Conclusions: ①As tight correlations existed between the expressions of Tiam 1, Rac 1 and the pathobiological behavior of gastric cancer, both of them maybe acted as reference indexes for judging the invasive and metastatic state of gastric cancer and forecasting the prognostic of patients. ②Positive correlations existed between the expressive intensity of Tiam 1 and the invasive and metastatic potentials of gastric cancer cells. ③Expressive restraint of Tiam 1 gene could directly result in reorganization of cytoskeleton and impair the invasive and metastatic potentials of gastric cancer cells both in vitro and in vivo. ④Based on our studys and other researchers'reports, the effects of Tiam 1 on the invasion and metastasis of gastric cancer cells, not only exhibited in changes of cellular morphology, but also originated from such cellular biological fundations as the activation of Rac 1 and the up-regulated synthesis of 67kDa LNR, NF-κB or MMP-2 / -9 proteins, which were induced or promoted by Tiam 1.