WNT4 REGULATES THE EFFECTS OF FORKHEAD BOX N1 ON THE APOPTOSIS OF THYMOMA CELLS THROUGH THE WNT–C-JUN N-TERMINAL KINASE SIGNALLING PATHWAY

Xiaowei He*, Yan Shi**, Xiaoniu Dong*, Ni Zhang***, #

*Department of Surgical Ward 2, Hangzhou Fuyang Hospital of Traditional Chinese Medicine, Hangzhou, PR China - **Department of Operating Room, Hangzhou Fuyang Hospital of Traditional Chinese Medicine, Hangzhou, PR China - ***Department of Surgical Ward 1, Hangzhou Fuyang Hospital of Traditional Chinese Medicine, Hangzhou, PR China

Objective: We sought to analyse the effects of Wnt4 gene interference on apoptosis and to explore whether the Wnt4 gene plays a role in regulating forkhead box N1 (FOXN1) through the Wnt4–c-Jun N-terminal kinase (JNK) signalling pathway. 

Methods: Wnt4 short hairpin RNA (shRNA)–interfering plasmids of four interfering targets of the Wnt4 gene were constructed. Contents of the blank, TR001 plasmid, and recombinant Wnt4-3–interfering plasmid groups were transformed into thymoma cells and modified with Lipofectamine™ 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Cell morphology observations were made under an inverted microscope, while cell apoptosis was analysed by flow cytometry. The effects of Wnt4-interfering plasmid on the expressions of Wnt4 and JNK protein were detected by western blot assay, and the effects of the Wnt4 gene on the expression of JNK gene after shRNA interference was detected by polymerase chain reaction (PCR) assay. The expressions of Wnt4 and FOXN1 messenger RNA in human thymoma tissue were also detected by PCR technology. Gene expression correlation was analysed by Spearman rank correlation analysis.

Results: The morphology of cells in the TR001 transfection group and Wnt4-3–interfering plasmid group showed changes, with chromatin aggregation, apoptotic bodies, and cell-membrane foaming observed. As compared with in the blank group, the apoptosis rate in the Wnt4-3–interfering plasmid group after cell transfection was significantly higher (P<0.05). There was no significant difference in the apoptosis rate of the Lipo2000 group and the TR001 group aa compared with the control (blank) group (P > 0.05). The expression levels of Wnt4 and JNK protein in the blank group and TR001 transfection group were higher than those in the Wnt4-3–interfering plasmid group, while the expression levels of Wnt4 and JNK protein in the Wnt4-3–interfering plasmid group were lower. Also, the expression level of the JNK gene after shRNA interference in the Wnt4 gene was significantly lower, which was consistent with the Wnt4 gene expression (P<0.05). Finally, the expressions of Wnt4 and FOXN1 messenger RNA in thymoma tissue were positively correlated (r≥1.012; P<0.01).

Conclusion: Wnt4-3–interfering plasmid can effectively inhibit the expression of the Wnt4 gene in thymoma cells. Interfering with the Wnt4 and FOXN1 genes in thymoma cells can promote apoptosis, which may be related to the Wnt4-JNK signalling pathway.