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

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,25μmol/L组、50μmol/L组和100μmol/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25μmol/L组、50μmol/L组和100μmol/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。

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 ofβ-catenin,TCF-4,caspase-3,and livin were measured by quantitative real-time PCR,while the expression levels ofβ-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 25μmol/L group,50μmol/L group,and 100μmol/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 ofβ-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 25μmol/L group,50μmol/L group,and100μmol/L group,the relative expression levels ofβ-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),respectively;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 ofβ-catenin/TCF-4 in renal cancer cells may lead to the transcriptional repression of livin and the up-regulation of caspase-3 expression,thereby inducing apoptosis of renal cancer cells.