| BackgroundGastric cancer is one of the malignant tumors which have high incidences in China, the death caused by which are mostly results from metastasis. Therefore, it is needed to further explore the mechanism of metastasis. TGF-β is such an interesting protein that in the early stage, it can suppress tumor growth but promote tumor invasion and metastasis in the late stage. Our previous research had shown that exogenous TGF-β significantly hastened the invasion and metastasis of gastric cancer cells. However, the mechanism of the opposite function has not been clarified. Smad3is a critical molecule in TGF-β signal pathway, whose carboxyl terminal and joining regions can both be phosphorylated (p-smad3C and p-smad3L as the phosphorylated product). Studies has shown that the role of TGF-β in tumor growth inhibition is mediated by the former, and its role in tumor invasion and metastasis promotion is mediated by the latter. Fascin, a cytoskeleton protein, which rarely expresses in normal epithelium, has been found overexpressed in various tumors including gastric cancer and highly related to tumor invasion and lymphatic metastasis. The purpose of this study is to investigate the effect of p-smad3L in the expression of fascin in gastric cancer cells induced by TGF-β, and further lay a foundation for elucidating the molecular mechanisms of TGF-β pathway in gastric cancer progression.Methods(1) The expression of fascin and growth of pseudopodia of gastric cancer cells were tested by immunofluorescence before and after TGF-β1treatment.(2) The expression of fascin was detected by RT-PCR and western blotting before and after TGF-β treatment at different points of time.(3) By conduction of smad3siRNA, the expression of fascin induced by TGF-β in gastric cancer cells was measured by RT-PCR and western blotting.(4) The phosphorylation of s204, s208and s213in the joining regions of smad3were evaluated by western blotting after TGF-β1treatment at different points of time.(5) Fascin promoter reported gene plasmid was constructed and its activity was assessed after the treatment of TGF-β1and different smad3mutants.Results(1) The number of pseudopodia increased obviously after the treatment of TGF-β1in the gastric cancer cell lines MKN45and AGS.(2) The strengthened expression and evident time regularity of fascin were seen in the gastric cancer cell lines MKN45, BGC823and AGS after TGF-β1treatment. There was a delay for0.5-2h and a peak at2-4h of the increasing expression of its mRNA. The expression of its protein reached the peak in4-8h after a delay period for0.5-2h, and then maintained4-12h before gradually weakened.(3) The expression of smad3mRNA and protein in smad3siRNA transfection group were down-regulated significantly, and so did the expression of fascin induced by TGF-β1.(4) The phosphorylation of s204, s208and s213in the joining regions of smad3was boosted in cell lines MKN45, BGC823and AGS after TGF-β1treatment. A time rule that protein expression peaked at1-4h after a delay period for0.5-2h and maintain for2-4h before gradually weakened.(5) The fascin promoter reported gene plasmid pGL3-FSCN1-Luc (-1375～-+81) were successfully constructed. The activity of fascinl-Luc was significantly higher in TGF-β1treated group (smad3WT, smad3EPSM and smad33A) than those without TGF-β1treatment in MKN45and BGC823(P<0.05). Among them, the activity of fascinl-Luc in the smad3WT transfection group was stronger than TGF-β1treatment group.Conclusions(1) The growth of pseudopodia of gastric cancer cells can be promoted by TGF-β1.(2) The expression of fascin in gastric cancer cells can be induced by TGF-β1with a evident time regularity. The activity of fascin can be enhanced by TGF-β1through smad3.(3) The phosphorylation of s204, s208and s213in the joining regions of smad3can be strengthened by TGF-β1with a time regularity in accordance with that of fascin expression.(4) The induction of fascin expression by TGF-β1depends on smad3, and the p-smad3L might play a crucial role in this process. |
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