ILK调控PLK1的表达对膀胱癌细胞恶性生物学行为的影响

Effect of ILK regulating expression of PLK1 on malignant biological behaviors of bladder cancer cells

目的探讨整合素连接激酶(integrin linked kinase,ILK)调控polo样激酶1(polo-like kinase 1,PLK1)的表达对膀胱癌细胞恶性生物学行为的影响。方法将构建好的针对ILK基因的过表达质粒在脂质体介导下稳定转染人膀胱癌5637细胞,并命名为5637/ILK组,同时设转染空质粒的5637细胞作为阴性对照,并命名为5637/NC组;运用Western blot分别检测5637/ILK和5637/NC组中ILK的表达。再将低表达PLK1的PLK1 siRNA质粒稳定转染过表达ILK的膀胱癌5637/ILK细胞,同时设未转染质粒的5637/ILK细胞为阴性对照,并将实验分为5637/ILK+PLK1 siRNA组和5637/ILK组。运用Western blot分别检测两组细胞中PLK1的表达;TUNEL细胞凋亡检测试剂盒检测两组细胞的凋亡;划痕实验检测两组细胞迁移能力;运用流式细胞仪检测两组细胞中活性氧(ROS)的含量。结果Western blot结果显示,5637/ILK组ILK蛋白表达较5637/NC组明显增高(P<0.05);PLK1蛋白的表达在5637/ILK+PLK1 siRNA组明显低于5637/ILK组(P<0.05)。TUNEL检测细胞凋亡结果显示,与5637/ILK组相比,5637/ILK+PLK1 siRNA组细胞的凋亡发生率显著增加(18.65%±1.26%vs 66.38%±0.73%,P<0.05)。划痕实验结果显示,5637/ILK+PLK1 siRNA组细胞在24 h内迁移距离小于5637/ILK组[(953.73±95.24)μm vs(1771.47±92.86)μm],差异具有统计学意义(P<0.05)。ROS实验显示,5637/ILK+PLK1 siRNA组细胞ROS荧光强度显著低于5637/ILK组,差异具有统计学意义(t=11.62,P<0.05)。结论下调PLK1的表达水平可使过表达ILK的膀胱癌5637/ILK细胞的凋亡明显增加,迁移能力显著降低,细胞内氧化应激状态减低,揭示ILK过表达所诱导的促癌效应很可能是通过对PLK1的激活作用来实现的。

Objective To investigate the effect of integrin linked kinase(ILK)regulating the expression of polo like kinase 1(PLK1)on the malignant biological behaviors of bladder cancer cells.Methods The human bladder cancer 5637 cells were transfected with overexpression ILK plasmid in 5637/ILK group,and empty plasmid in 5637/NC group.Western blot was used to detect the expression of ILK in 5637/ILK group and 5637/NC group.Then,the PLK1 siRNA plasmid with low PLK1 expression was stably transfected into the bladder cancer 5637/ILK cells as 5637/ILK+PLK1 siRNA group,meanwhile,the 5637/ILK cells were not transfected with plasmid as negative control(5637/ILK group).Western blot was used to detect the expression of PLK1.TUNEL cell apoptosis detection kit was used to detect the apoptosis.The scratch test was used to measure the ability of cell migration.Flow cytometry was used to detect the content of reactive oxygen species(ROS).Results Western blot showed that the expression of ILK protein in 5637/ILK group was significantly higher than that in 5637/NC group(P<0.05).The expression of PLK1 protein in 5637/ILK+PLK1 siRNA group was significantly lower than that in 5637/ILK group(P<0.05).The apoptosis rate in 5637/ILK+PLK1siRNA group was signi-ficantly higher than that in 5637/ILK group(66.38%±0.73%vs 18.65%±1.26%,P<0.05).Scratch test showed that the migration distance within 24 h was less in 5637/ILK+PLK1 siRNA group than in 5637/ILK group[(953.73±95.24)μm vs(1771.47±92.86)μm,P<0.05].ROS fluorescence intensity in 5637/ILK+PLK1 siRNA group was significantly lower than that in 5637/ILK group(t=11.62,P<0.05).Conclusion The lower expression of PLK1 can significantly increase the apoptosis of bladder cancer 5637 cells overexpressing ILK,decrease the migration ability and decrease the oxidative stress status,which suggests that the oncogenic effect induced by ILK overexpression may be realized through the activation of PLK1.