Abstract:

Objective To discuss the effect of gene silencing of targeting protein for Xenopus kinesin like protein 2 （TPX2） on the chemosensitivity of the resistant bladder cancer cell line T24/cisplatin（DDP）， and to clarify the mechanism. Methods The DDP-resistant cell line T24/DDP was established by DDP concentration gradient induction method， and the cells were divided into T24 cell group and T24/DDP cell group. MTT method was used to detect the proliferation activities of the cells in various groups； the resistance index （RI） was calculated based on the half maximal inhibitory concentration （IC₅₀） value；real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods were used to detect the expression levels of TPX2 mRNA and protein in the cells in various groups； small interfering RNA （siRNA） was used to silence the TPX2 gene expression in the T24/DDP cells.The cells were divided into blank control group， negative control siRNA （si-NC） group， TPX2 silenced （si-TPX2） group， si-NC+DDP （2 mg·L^-1 DDP） group，and si-TPX2+DDP （2 mg·L^-1 DDP） group. RT-qPCR and Western blotting methods were used to detect the expression levels of TPX2 mRNA and protein in the cells in various groups； MTT method was used to detect the proliferation activity of the cells in various groups； flow cytometry was used to detect the apoptotic rates of the cells and percentages of the cells at G₂/M phase in various groups； Transwell chamber assay was used to detect the numbers of migration and invasion cells； Western blotting method was used to detect the expression levels of Wnt/β-catenin signaling pathway-related proteins such as β-catenin， P-glycoprotein （P-gp）， zinc finger protein transcription factor 1 （Snail1）， and Survivin proteins in the cells in various groups. Results The resistant bladder cancer cell line T24/DDP was successfully established with the RI value of 8.76. Compared with T24 cell group， the expression levels of TPX2 mRNA and protein in the T24/DDP cells were significantly increased （P<0.01）. Compared with blank control group and si-NC group， the expression levels of TPX2 mRNA and protein in the T24/DDP cells in si-TPX2 group were significantly decreased （P<0.01）， and the IC₅₀ value of DDP was significantly decreased （P<0.01）. Compared with si-NC group， the apoptotic rate of the cells and the percentage of the cells at G₂/M phase in si-TPX2 group was significantly increased （P<0.01）， and the numbers of migration and invasion cells were significantly decreased （P<0.01）， and the expression levels of β-catenin， P-gp， Snail1， and Survivin proteins in the T254/DDP cells were also significantly decreased （P<0.01）. Compared with si-NC+DDP group， the apoptotic rate of the cells and percentage of the cells at G₂/M phase in si-TPX2+DDP group were significantly increased （P<0.01）， and the numbers of migration and invasion cells were significantly decreased （P<0.01）， and the expression levels of β-catenin， P-gp， Snail1， and Survivin proteins in the T24/DDP cells were significantly decreased (P<0.01). Conclusion Gene silencing of TPX2 enhances the chemosensitivity of the resistant bladder cancer cell line T24/DDP to DDP by inhibiting the Wnt/β-catenin signaling pathway.

Key words: Targeting protein for Xenopus kinesinlike protein 2, Bladder neoplasm, Cisplatin, Chemotherapy sensitivity, Wnt/β-catenin signaling pathway