| Background and Objectives Wnt signaling pathway was one of the most important pathways playing key roles in vertebrate embryonic development, involved in cell differentiation, apoptosis, proliferation and cellular localization control process. Previous studies had demonstrated that aberrant activation of the Wnt signaling pathway was associated with the occurrence and development of a wide variety of tumor closely. T cell factor 4 (TCF-4) was a significant regulator in Wnt signaling pathway. The silence or over-expression of TCF-4 could cause Wnt signaling dysregulation. This study intended to explore the expression of T cell factor 4 (TCF-4) and the function of proliferation, senescence and apoptosis in gastric cancer cells.Materials and Methods (1) Human gastric cancer cell lines MGC803, SGC7901, HSC44-PE,44As3, MKN45, NCI-N87 and immortalized human gastric epithelial cell line GES-1 were cultured and the differential expression of TCF-4 among them were detected by western blot analysis. (2) The lowest and highest one were selected and transfected with TCF-4 expression and dominant negative TCF-4 plasmid respectively. Knock-in TCF-4 SGC/TCF-4 cell line and knock-down TCF-4 N87/dnTCF-4, 44As3/dnTCF-4 cell lines were screened by puromycin (PM) in accordance with instructions. (3) In order to research the influence on cell proliferation and apoptosis of TCF-4 in gastric cancer cells, two control groups (NCI-N87 VS n87/dnTCF-4, SGC7901 VS SGC/TCF-4) were established and detected by cell proliferation assay and RTCA. (4) Gastric cancer cell lines NCI-N87, N87/dnTCF-4, SGC7901, SGC/TCF-4 were inoculated in 35mm dishes and each dish included 5×103 cells. Senescence P-Galactosidase Staining Kit was used to stain after 48 h and the positive cells were counted under microscope. (5) Meanwhile, the models of nude-mice bearing tumor were established to detect the effect of TCF-4 on growth in vivo. The volumes were determined twice a week to observe the influence of TCF-4 on growth in otransplanted tumor.Results (1) The expression of TCF-4 in gastric cancer cells were higher than the control GES-1, the highest one was NCI-N87, followed by 44As3, HSC44-PE, MGC803, MKN45 and SGC7901. (2) Using western blot to verify the transfection efficiency, the expression of TCF-4 in SGC/TCF-4 was higher than in SGC7901, the same were true in the other two control groups (NCI-N87 VS N87/dnTCF-4,44As3 VS 44As3/dnTCF-4). (3) Cell proliferation analysis and RTCA showed that N87/dnTCF-4 cell proliferation was significantly lower than NCI-N87, SGC/TCF-4 cell proliferation was higher than SGC7901 distinctly. (4) Cell Senescence β-Galactosidase Stain analysis revealed that the ratios of positive staining cell in NCI-N87, N87/dnTCF-4, SGC7901 and SGC/TCF-4 were 7.7%,18.7%,8.2% and 1.2% respectively. (5) Nude-mice tumorigenicity assay revealed that tumor volume of SGC7901 was smaller, NCI-N87 was larger than control.Conclusion (1) Comparing with control immortalized human gastric epithelial cell, the expression of TCF-4 in gastric cancer cell was higher. This result indicated that TCF-4 had a certain correlation with the occurrence and development of gastric cancer. (2) TCF-4 knock-in cell line SGC/TCF-4, and TCF-4 knock-down cell lines N87/dnTCF-4,44As3 were constructed through plasmid transfection technology successfully. The nude-mice models of gastric cancer with TCF-4 over-expression and lower-espression was established well. The experiments in vivo and in vitro were further prove that TCF-4 could promote the proliferation, inhibit the senescence and apoptosis of gastric cancer cells. |
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