目的：探讨敲减中心体相关激酶2（never in mitosis A-related kinase 2，NEK2）对结直肠癌细胞5-FU化疗敏感性的影响及其可能的机制。方法：采用 qPCR 和 Western blotting 检测结直肠癌细胞中 NEK2 mRNA 及蛋白的表达水平。构建针对NEK2基因的小干扰RNA（siRNA）并转染至结直肠癌细胞HCT116及SW620，实验分为阳性干扰组1（转染NEK2 siRNA1）、阳性干扰组2（转染NEK2 siRNA2）和阴性对照组（转染si-NC），均用5-FU处理。采用CCK-8实验、V-FICT/PI Annexin双染色流式细胞术实验观察敲减NEK2基因对5-FU作用下结肠癌细胞的增殖、周期分布及凋亡的影响，采用Western blotting检测敲减NEK2基因对5-FU作用下结直肠癌细胞内Wnt/β-catenin信号通路相关蛋白表达的影响。结果：NEK2蛋白及mRNA在结直肠癌细胞HCT116、SW620中均呈高表达（P<0.05或P<0.01），转染NEK2 siRNA可高效抑制HCT116、SW620细胞中NEK2蛋白及mRNA表达（均P<0.01）。经不同浓度5-FU作用后，阳性干扰组1和阳性干扰组2的细胞存活率和IC50均显著低于阴性对照组（均P<0.01），细胞发生G0/G1期阻滞且凋亡率显著升高（均 P<0.01），胞核 β-catenin、c-myc 和 cyclin D1 表达水平显著下降而胞质β-catenin表达水平升高（均P<0.01）。结论：敲减NEK2基因可有效提高人结直肠癌细胞对5-FU的化疗敏感性，该作用可能是通过调控Wnt/β-catenin信号通路相关蛋白表达来实现的。

Objective：To investigate the effects of RNA interfering NEK2 (NIMA-related kinase 2) gene on the sensitivity of colorectal carcinoma (CRC) cell lines to 5-fluorouracil (5-FU) and the possible mechanisms. Methods: The mRNA and protein expressions of NEK2 in CRC cell lines were detected by quantitative polymerase chain reaction (qPCR) and Western blotting assay,respectively. siRNAs targeting NEK2 were constructed and transfected into CRC HCT116 and SW620 cells. The experiments were divided into positive interference group 1 (transfection with NEK2 siRNA1), positive interference group 2 (transfection with NEK2 siRNA2) and negative control group (transfection with si-NC), all the CRC cells were treated with 5-FU. CCK-8 assay, Flow cytometry and V-FICT/PI Annexin staining analysis were used to observe the effects of NEK2 gene knockdown on proliferation, cell cycle distribution and apoptosis of CRC cells under 5-FU treatment. Western blotting was used to detect the effects of NEK2 gene knockdown on the expression of Wnt/β-catenin signaling pathway related proteins in CRC cells under 5-FU treatment. Results: NEK2 was highly expressed in CRC HCT116 and SW620 cells at both protein and mRNA levels (all P<0.05). siRNA NEK2 transfection could effectively inhibit the protein and mRNA expressions of NEK2 in HCT116 and SW620 cells (all P<0.01). After treatment with various concentrations of 5-FU, the cell survival rate and IC50, as well as the expression levels of β-catenin (cytoblasts), C-MYC and Cyclin D1,in the positive interference group 1 and 2 were significantly decreased, while cell cycle blockage at G0/G1 phase, the apoptosis rate and the expression level of β-catenin (cytoplasm) were significantly increased (all P<0.01), as compared with the negative control group (all P<0.01). Conclusion: Silencing NEK2 gene can effectively improve the sensitivity of human colorectal cancer cells to 5-FU, which may be achieved by regulating the expression of Wnt/β-catenin signaling pathway related proteins.

河南省医学科技攻关计划资助项目（No. 201702246）