Survivin对食管癌细胞Tak1、NF-κB表达调控以及细胞周期和凋亡的影响

Regulatory effects of Survivin on Tak1 and NF-κB in esophageal carcinoma cells and its effects on cell cycle and apoptosis

目的探讨存活蛋白(Survivin)对食管癌细胞转化生长因子β激活激酶1(Tak1)、核因子κB(NF-κB)表达调控以及细胞周期和凋亡的影响。方法常规体外培养人食管癌细胞Eca109,随机分为空白对照组、Survivin-siRNA组、Survivin-siRNA空载体组、Survivin过表达组、Survivin过表达空载体组、YM155组,Survivin-siRNA组与Survivin-siRNA空载体组分别转染Survivin siRNA载体、Survivin siRNA空载体,Survivin过表达组与Survivin过表达空载体组分别转染Survivin过表达载体、Survivin过表达空载体,转染6 h,更换新鲜培养基继续培养24 h,收集细胞。空白对照组不予转染,YM155组加入Survivin抑制剂YM155,于各转染组同期收集细胞。采用Western blotting法检测Survivin、磷酸化Tak1(p-Tak1)、NF-κB p65表达,采用流式细胞术检测细胞周期分布和细胞凋亡率。结果与空白对照组比较,Survivin过表达组Survivin相对表达量显著升高,而Survivin-siRNA组和YM155组Survivin相对表达量显著降低(P均<0.05)。与空白对照组比较,各转染组及YM155组p-Tak1、NF-κB相对表达量无显著变化(P均>0.05)。与空白对照组比较,YM155组G_(2)期细胞所占比例显著升高(P<0.05),S期细胞所占比例显著降低(P<0.05),G_(1)期细胞所占比例无显著变化(P>0.05);与空白对照组比较,各转染组G_(1)、G_(2)、S期细胞所占比例均无显著变化(P均>0.05)。与空白对照组比较,Survivin-siRNA组早期凋亡率和总凋亡率显著升高(P均<0.05),晚期凋亡率无显著变化(P>0.05);与空白对照组比较,其他转染组和YM155组早期凋亡率、晚期凋亡率、总凋亡率均无显著变化(P均>0.05)。结论Survivin能够参与食管癌细胞周期分布和凋亡调控,但其作用途径并非通过调控Tak1、NF-κB表达实现。

Objective To investigate the regulatory effects of Survival protein(Survivin)on the expression of transforming growth factorβ-activated kinase 1(Tak1)and nuclear factorκB(NF-κB),cell cycle,and apoptosis of esophageal cancer cells.Methods Human esophageal carcinoma cells Eca109 were cultured in vitro and divided into the blank control group,Survivin-siRNA group,Survivin-siRNA empty vector group,Survivin overexpression group,Survivin overexpression empty vector group,and YM155 group.Cells in the Survivin-siRNA and Survivin-siRNA empty vector groups were transfected with Survivin siRNA vector or empty vector of Survivin siRNA,respectively.Cells in the Survivin overexpression group and the Survivin overexpression empty vector group were transfected with the overexpression vector of Survivin or the overexpression empty vector of Survivin for 6 h,respectively,and then they were cultured in a fresh medium for 24 h to collect the cells.Cells in the blank control group were not transfected,and cells in the YM155 group were added with Survivin inhibitor YM155.After that,we collected the cells in each transfection group simultaneously.The expression levels of Survivin,phosphorylated Tak1(p-Tak1),and NF-κB p65 were detected by Western blotting,and the cell cycle distribution and apoptosis rate were detected by flow cytometry.Results Compared with the blank control group,the relative expression of Survivin significantly increased in the Survivin overexpression group but significantly decreased in the Survivin-siRNA group and YM155 group(all P<0.05).Compared with the blank control group,the expression levels of p-TAK1 and NF-κB protein in each transfection group and YM155 group had no significant changes(all P>0.05).Compared with the blank control group,the proportion of cells in G_(2) phase significantly increased(P<0.05),the proportion of cells in S phase significantly decreased(P<0.05),and the proportion of cells in the G_(1) phase had no significant change in the YM155 group(all P>0.05).Compared with the blank control group,there were no significant changes in the proportions of cells in the G_(1),G_(2),and S phases in each transfection group(all P>0.05).Compared with the blank control group,the early apoptosis rate and total apoptosis rate of the Survivin-siRNA group increased(both P<0.05),but the late apoptosis rate had no significant change(P>0.05).Compared with the blank control group,there were no significant changes in the early apoptosis rate,late apoptosis rate,or total apoptosis rate in the other transfection groups and the YM155 group(all P>0.05).Conclusion Survivin expression is involved in regulating cell cycle and apoptosis in esophageal cancer cells,but its effect is not implemented by regulating Tak1 and NF-κB.