| Objective. To investigate the effect of transcription factor ZBTB14 on Wnt /β-catenin signal in colon cancer SW480 cells and explore its mechanism.Methods.1. Constructed a vector plasmid which could express ZBTB14. The molecular weigth of expressed ZBTB14 proteins was confirmed by Western blot analysis.TOPflash assay was used to detect the activity of Wnt/β-catenin signal in HEK293T cell after the transfection of ZBTB14, Dvl, LRP, β-catenin and P-catenin-lefl. The FOPflash was used as a negative control.2. Western blot analysis was performed to detect the expression of β-catenin and non-phosphorylated-β-catenin proteins in HEK293T cells and SW480 cells after the transfection of ZBTB14 plasmid. Tubulin was used as internal control.3. Immunofluorescence was used to detect the expression of β-catenin and non-phosphorylated-β-catenin proteins in HEK293T cells and SW480 cells.4. Real-time PCR was performed to measure the relative β-catenin, TIAM1 and Cyclin D1 mRNA levels in HEK293T cells. Samples were normalized to GAPDH expression.Results1. Wnt3a, Dvl, LRP and P-catenin are able to activate Wnt/β-catenin signal in HEK293T cells, but the activation effect can be inhibited by ZBTB14 proteins.While ZBTB14 proteins can’t inhibit the activation effect of β-catenin-lefl.2. Western blot analysis and immunofluorescence show that ZBTB14 proteins reduce the amount of P-catenin and non-phosphorylated-β-catenin proteins both in HEK293T cells and SW480 cells.3. Overexpression of ZBTB14 may decrease the synthesis of β-catenin proteins by reducing P-catenin mRNA level both in HEK293T and SW480 cells.4. The inhibition of Wnt/β-catenin signal will lead to the decreasing expression of Wnt/β-catenin signal target genes, such as TIAM1 and Cyclin D1.Conclusions Transcription factor ZBTB14 inhibits Wnt/β-catenin signal in SW480 cells by reducing the expression of β-catenin proteins. Therefore ZBTB14 is a potential tumor inhibiting factor. |
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