Abstract
BACKGROUND: To explore the impact of thymoma cell apoptosis and the signal pathway that Wnt4 gene works through in thymoma cells after interfering Wnt4 gene by using shRNA. METHODS: Here we construct four shRNA-Wnt4 interference plasmids which were directed at Wnt4 gene. The blank plasmid, TR001 plasmid and recombinant plasmids were transfected into thymoma cells. The expression of Wnt4 was detected by RT-PCR meanwhile selected the best Wnt4 interference plasmid for subsequent experiments. The thymoma cells were divided into four groups: control group, lipo2000 group; transfected TR001 plasmid, transfected interference plasmid. The impact of apoptosis after down Wnt4 gene was detected by Wright’s-Giemsa’s mixed stain, Hoechst-33342/PI fluorescence stain, flow cytometry and Western blot detection of Caspase-3 and cleavage fragments. At the same time the thymoma cells were divided into three groups: control group, transfected TR001 plasmid, transfected interference plasmid. The expression of Wnt4, JNK and β-catenin were detected by RT-PCR and Western blot. RESULTS: We have compared with the corresponding control groups, the shRNA-Wnt4-3 plasmid is the best Wnt4 interference plasmid for subsequent experiments. The ratio of inhibition was 52.37% (P<0.05). Furthermore, the apoptosis rate was significantly increased in shRNA-Wnt4-3 plasmid group (14.70±0.62, P<0.001). Lastly, when down Wnt4 the expression of JNK was significantly down-regulated in thymoma cells (P<0.01). CONCLUSIONS: We suggest that the shRNA-Wnt4-3 plasmid which we constructed can significantly interference the expression of Wnt4 gene in thymoma cell. Through interfering Wnt4 gene can promote the apoptosis rate of the human thymoma cell. This appearance indicates that the aberrant expression of Wnt4 gene may be one of the mechanisms of occurrence and development of thymoma. After interference Wnt4 gene by using shRNA, the expression of JNK gene is significantly reduced. This appearance indicates that Wnt4 gene is correlated with the expression of JNK gene in thymoma cells. And this work may depend on the non-canonical Wnt/JNK signaling pathway.