METHODS: The expression of TOPK was detected in CRC tissues by immunohistochemistry, and the effect of TOPK knockdown was detected in CRC cells by Western blotting. CCK-8 and clonogenic assays were used to detect the growth and clonogenic ability of CRC cells after TOPK knockdown combined with radiotherapy in CRC cells. Furthermore, proteomic analysis showed that the phosphorylation of TOPK downstream proteins changed after radiotherapy. DNA damage was detected by the comet assay. Changes in the DNA damage response signaling pathway were analyzed by Western blotting, and apoptosis was detected by flow cytometry.
RESULTS: The expression of TOPK was significantly greater in CRC tissues at grades 2-4 than in those at grade 1. After irradiation, CRC cells with genetically silenced TOPK had shorter comet tails and reduced expression levels of DNA damage response-associated proteins, including phospho-cyclin-dependent kinase 1 (p-CDK1), phospho-ataxia telangiectasia-mutated (p-ATM), poly ADP-ribose polymerase (PARP), and meiotic recombination 11 homolog 1 (MRE11).
CONCLUSIONS: TOPK was overexpressed in patients with moderately to poorly differentiated CRC. Moreover, TOPK knockdown significantly enhanced the radiosensitivity of CRC cells by reducing the DNA damage response.
方法:免疫组化法检测CRC组织中TOPK的表达,通过Western印迹在CRC细胞中检测到TOPK敲低的作用。采用CCK-8和克隆形成试验检测TOPK敲除联合放疗后CRC细胞的生长和克隆形成能力。此外,蛋白质组分析显示放疗后TOPK下游蛋白磷酸化发生改变。通过彗星试验检测DNA损伤。通过蛋白质印迹分析DNA损伤反应信号通路的变化,流式细胞术检测细胞凋亡。
结果:TOPK在2-4级CRC组织中的表达明显高于1级。辐照后,具有遗传沉默的TOPK的CRC细胞具有较短的彗星尾巴和降低的DNA损伤反应相关蛋白的表达水平,包括磷酸化细胞周期蛋白依赖性激酶1(p-CDK1),磷酸化共济失调毛细血管扩张症突变(p-ATM),聚ADP-核糖聚合酶(PARP),和减数分裂重组11同源物1(MRE11)。
结论:TOPK在中度至低分化CRC患者中过度表达。此外,TOPK敲除通过降低DNA损伤反应而显著增强CRC细胞的放射敏感性。