Wnt/β-catenin/TCF-4信号通路调节livin表达在肾癌细胞增殖凋亡中的研究

Role of the Wnt/β-catenin/TCF-4 signaling pathway in the regulation of livin expression in proliferation and apoptosis of renal cancer cells

摘要:

目的：研究人肾癌细胞系786-0中Wnt/β-catenin/TCF-4通路与livin的关系，探索Wnt/β-catenin/TCF-4通路在肾癌细胞中的凋亡调节机制。方法：不同浓度吲哚美辛处理肾癌786-0细胞，CCK-8法检测各组细胞活性；流式细胞术检测各组细胞凋亡改变；荧光定量PCR检测β-catenin、TCF-4、caspase-3及livin转录水平变化；Western blot检测β-catenin、caspase-3及livin蛋白表达水平变化。结果：随吲哚美辛浓度增加，细胞活力降低，呈剂量-效应关系（P=0.000）。流式结果显示伴随吲哚美辛浓度升高，细胞凋亡逐渐增加，25 μmol/L组、50 μmol/L组和100 μmol/L组凋亡率分别为（32.07±1.01）%、（40.03±1.95）%和（72.33±0.02）%，与对照组比较差异存在统计学意义（P=0.000，P=0.002，P=0.000）。Real-time PCR结果显示，β-catenin、TCF-4和livin相对表达水平均呈下降趋势（P=0.002，P=0.000，P=0.000），而caspase-3相对表达呈明显上升趋势（P=0.001）。Western blot分析显示在25 μmol/L组、50 μmol/L组和100 μmol/L组中β-catenin相对表达量分别为0.568±0.020（P=0.001）、0.396±0.030（P=0.000）、0.142±0.038（P=0.000）；livin相对表达量分别为0.139±0.016（P=0.005）、0.050±0.006（P=0.000）、0.011±0.001（P=0.000）；而caspase-3相对表达量分别为0.278±0.035（P=0.046）、0.396±0.071（P=0.000）、0.518±0.015（P=0.000）。结论：吲哚美辛介导的肾癌细胞中β-catenin/TCF-4降解可能导致livin的转录抑制和caspase-3表达水平的上调，进而诱导肾癌细胞凋亡。

Abstract:

Objective：To investigate the relationship between the Wnt/β-catenin/TCF-4 pathway and livin expression in human renal cancer cell line 786-0，and to explore the apoptosis regulation mechanism of this pathway. Methods：786-0 cells were treated with different concentrations of indomethacin. Cell viability was then determined by CCK-8 assay. Flow cytometry was used to determine apoptosis change. The transcriptional changes of β-catenin，TCF-4，caspase-3，and livin were measured by quantitative real-time PCR，while the expression levels of β-catenin，caspase-3，and livin were measured by Western blot. Results：With the increase in the concentration of indomethacin，the cell viability decreased in a dose-response manner（P=0.000）. Flow cytometry showed that the apoptosis rate increased with the increase in the concentration of indomethacin. The apoptosis rates of the 25 μmol/L group，50 μmol/L group，and 100 μmol/L group were （32.07±1.01）%，（40.03±1.95）%，and （72.33±0.02）%，respectively，all showing significant differences compared with those of the control group（P=0.000，0.002，and 0.000，respectively）. Real-time PCR showed that the relative expression levels of β-catenin，TCF-4，and livin decreased significantly（P=0.002，0.000，and 0.000，respectively），while the level of caspase-3 increased significantly（P=0.001）. Western blot revealed that，in the 25 μmol/L group，50 μmol/L group，and 100 μmol/L group，the relative expression levels of β-catenin were 0.568±0.020（P=0.001），0.396±0.030（P=0.000），and 0.142±0.038（P=0.000），respectively；the levels of livin were 0.139±0.016（P=0.005），0.050±0.006（P=0.000），and 0.011±0.001（P=0.000），re－spectively；and the levels of caspase-3 were 0.278±0.035（P=0.046），0.396±0.071（P=0.000），and 0.518±0.015（P=0.000），respectively. Conclusion：Indomethacin-mediated degradation of β-catenin/TCF-4 in renal cancer cells may lead to the tran－scriptional repression of livin and the up-regulation of caspase-3 expression，thereby inducing apoptosis of renal cancer cells.