TPX2基因沉默对膀胱癌耐药细胞株T24/DDP顺铂化疗敏感性的增强作用及其机制

Enhancement effect of TPX2 gene silencing on chemosensitivity of bladder cancer cell line T24/DDP to cisplatin and its mechanism

摘要

目的 探讨爪蟾驱动蛋白样蛋白2靶蛋白（TPX2）基因沉默对膀胱癌耐药细胞株T24/顺铂（DDP）的化疗敏感性的影响，并阐明其作用机制。 方法 采用DDP浓度梯度刺激法建立DDP耐药细胞株T24/DDP，将细胞分为T24细胞组和T24/DDP细胞组。MTT法检测各组细胞增殖活性，并根据半数抑制浓度（IC₅₀）值计算耐药指数（RI）；实时荧光定量聚合酶链式反应（RT-qPCR）法和Western blotting法检测细胞中TPX2 mRNA及蛋白表达水平。通过小干扰RNA（siRNA）沉默T24/DDP细胞中TPX2基因表达，再将细胞分为空白对照组、阴性对照干扰（si-NC）组、TPX2沉默（si-TPX2）组、si-NC+DDP（2 mg·L^－1 DDP）组和si-TPX2+DDP（2 mg·L^－1 DDP）组。RT-qPCR法和Western blotting法检测转染后细胞中TPX2 mRNA及蛋白表达水平，MTT法检测各组细胞增殖活性，流式细胞术检测各组凋亡细胞率和细胞周期G₂/M期百分率，Transwell小室实验检测各组迁移细胞数和侵袭细胞数，Western blotting法检测各组细胞中Wnt/β-连环蛋白（β-catenin）信号通路相关蛋白β-catenin、P-糖蛋白（P-gp）、锌指蛋白转录因子1（Snail1）和Survivin蛋白表达水平。 结果 成功建立膀胱癌DDP耐药细胞株T24/DDP，RI值为8.76。与T24细胞组比较，T24/DDP细胞组中TPX2 mRNA和蛋白表达水平明显升高（P<0.01）。与空白对照组和si-NC组比较，si-TPX2组T24/DDP细胞中TPX2 mRNA和蛋白表达水平明显降低（P<0.01），且DDP的IC₅₀值明显降低（P<0.01）。与si-NC组比较，si-TPX2组T24/DDP细胞凋亡率和细胞周期G₂/M期百分率明显升高（P<0.01），迁移细胞数和侵袭细胞数明显降低（P<0.01），T24/DDP细胞中β-catenin、P-gp、Snail1和Survivin蛋白表达水平明显降低（P<0.01）；与si-NC+DDP组比较，si-TPX2+DDP组T24/DDP细胞凋亡率和细胞周期G₂/M期百分率明显升高（P<0.01），迁移细胞数和侵袭细胞数明显降低（P<0.01），T24/DDP细胞中β-catenin、P-gp、Snail1和Survivin蛋白表达水平明显降低（P<0.01）。 结论 TPX2基因沉默通过抑制Wnt/β-catenin信号通路增强膀胱癌耐药细胞株T24/DDP对DDP的化疗敏感性。

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.

关键词

爪蟾驱动蛋白样蛋白2靶蛋白 / 膀胱肿瘤 / 顺铂 / 化疗敏感性 / Wnt/β-catenin信号通路

Key words

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

Author summay

张 鹰（1987－），男，湖南省衡阳市人，医学硕士，主治医师，主要从事重症医学相关方面的研究。