WNK1在结直肠癌中的表达情况及对预后的影响

Expression of WNK1 in Colorectal Cancer and Its Influence on Prognosis

目的探讨WNK1在结直肠癌(CRC)中的表达情况及对预后的影响。方法采用免疫组织化学方法检测68例CRC患者中WNK1基因的表达水平。采用log-rank检验和Kaplan-Meier生存曲线分析WNK1表达水平与总生存期(OS)和无进展生存期(PFS)的关系。采用蛋白免疫印迹法对细胞的上皮和间质标志物进行分析,通过Transwell分析与划痕实验验证WNK1基因对CRC细胞迁移能力的影响。结果 CRC组织WNK1的表达阳性率[(55.9%(38/68)]高于癌旁组织[14.7%(10/68)],差异有统计学意义(P<0.05)。WNK1的表达与淋巴结转移和TNM分期相关(均P<0.05)。WNK1高表达CRC患者OS率和PFS率均低于WNK1低表达患者(均P<0.05)。经蛋白免疫印迹法证实WNK1在HCT116和HCT15细胞系中的表达均下调,HCT116细胞中灰度降低72%,HCT15细胞中灰度降低85%。在HCT116和HCT15细胞中,WNK1的下调可恢复上皮标志物钙黏附蛋白E的表达,并伴随间质标记物波形蛋白的丢失。Transwell结果显示,采用siRNA抑制WNK1基因后HCT116细胞中通过滤膜迁移至下室的细胞(57.00±4.36)少于转染空载体对照组(230.00±8.89),差异有统计学意义(P<0.05);HCT15细胞中通过滤膜迁移至下室的细胞(61.00±3.61)少于转染空载体对照组(241.00±5.52),差异有统计学意义(P<0.05)。划痕实验结果显示,在HCT116中下调WNK1基因后细胞48 h划痕愈合率(10.7%)低于转染空载体对照组(60.3%),差异有统计学意义(P<0.05);在HCT15中下调WNK1基因后细胞48 h划痕愈合率(7.8%)低于转染空载体对照组(45.8%),差异有统计学意义(P<0.05)。结论 WNK1在CRC中的表达显著高于正常结直肠组织,且WNK1高表达与CRC TNM分期及复发转移相关。抑制WNK1基因可抑制上皮细胞-间充质转换(EMT)和CRC细胞的迁移能力。

Objective To investigate the expression of WNK1 in colorectal cancer and its influence on prognosis.Methods The expressions of WNK1 gene in 68 patients with colorectal cancer were detected by immunohistochemistry.Log-rank test and Kaplan-Meier survival curve were used to analyze the relationships between the expression level of WNK1 and overall survival(OS)and progression-free survival(PFS).Western blotting was used to analyze epithelial and stromal markers of cells.The influence of WNK1 gene on the migration ability of CRC cells through Transwell analysis and scratch test was verified.Results The positive rate[(55.9%(38/68)]of WNK1 expression in CRC tissues was higher than that[14.7%(10/68)]in paracancer tissues,and the difference was statistically significant(P<0.05).The WNK1 expression was correlated with lymph node metastasis and TNM stage(both P<0.05).The OS rate and PFS rate of patients with high expression of WNK1 were both lower than those of patients with low expression of WNK1(both P<0.05).Western blotting showed that WNK1 was down regulated in HCT116 cell line and HCT15 cell line.The gray level of HCT116 cells decreased by 72%,and the gray level of HCT15 cells decreased by 85%.In HCT116 and HCT15 cells,down-regulation of WNK1 can restore the expression of E-cadherin(an epithelial marker),with the loss of vimentin(an interstitial marker).Transwell showed that the number of cells(57.00±4.36)in HCT116 cells that migrated to the lower chamber through the filter membrane after WNK1 gene was inhibited by siRNA was lower than that(230.00±8.89)in control group transfected with empty vector,and the difference was statistically significant(P<0.05).The number of cells(61.00±3.61)in HCT15 cells that migrated to the lower chamber through the filter membrane after WNK1 gene was inhibited by siRNA was lower than that(241.00±5.52)in control group transfected with empty vector,and the difference was statistically significant(P<0.05).Scratch test showed that the scratch healing rate in 48 hours(10.7%)in HCT116 after WNK1 gene was down regulated was lower than that(60.3%)in control group transfected with empty vector(P<0.05).The scratch healing rate in 48 hours(7.8%)in HCT15 after WNK1 gene was down regulated was lower than that(45.8%)in control group transfected with empty vector,and the difference was statistically significant(P<0.05).Conclusion The expression of WNK1 in CRC is significantly higher than that in normal colorectal tissues,and the high expression of WNK1 is correlated with TNM stage,recurrence and metastasis of CRC.Inhibition of WNK1 gene can inhibit epithelial-mesenchymal transition(EMT)and migration of CRC cells.