Objective To investigate the effect of IKKε in glioma cells on the regulation of Ecadherin expression and cell migration and invasion ability.Methods The IKKε overexpression vectors and shRNA-IKKε lentivirus were constructed.The TP483 glioma cell lines were transfected by the IKKε overexpression plasmid.The expression level of IKKε in glioma cells was upregulated.The U251 and U138 glioma cell lines were transfected by the shRNA-IKKε lentivirus,IKKε expression level in glioma cells was downregulated.The expression levels of IKKε and E-cadherin in cells were detected by RT-PCR and Western blot.The expression changes of IKKε and E-cadherin after transfecting were observed by using the immunofluorescence assay.The cell migration and invasion abilities were detected by using the scratch test and Transwell assay.Results Compared with the non-transfected group (TP483 control group),the expression level of IKKε protein increased (P =0.000),and the expression level of E-cadherin protein decreased (P =0.000) in the transfected IKKε expression plasmid group (TP483 treaed group).In the glioma cells transfected IKKε shRNA(the U251 treated group and the U138 treated group),the expression level of IKKε protein decreased significantly compared with the non-transfected group (the U251 treated group and the U138 treated group;all P ＜ 0.05).At the same time,the expression levels of E-cadherin protein in each treated group were all increased compared with the control group (P ＜ 0.05).The scratch repair rate of the TP483 treated group was higher than that of the control group (56.39 ± 0.93％ vs.46.43 ± 1.68％;P =0.035).After transfection of overexpression plasmid for 24 h,the number of TP483 cells passing through the filter membrane increased compared with the control group (55.8 ± 6.0 vs.24.6 ± 2.3;P =0.000).The scratch repair rates of the U251 and U138 treated groups decreased compared with their respective control groups (the U251 treated group:50.40 ± 1.09％,the U251 control group:87.29 ± 11.1 1％,P =0.043;the U138 treated group:48.20 ± 1.34％,the U138 control group:73.15 ± 6.62％,P =0.035);After transfectiou for 24 h,the number of cells passing through the filter membrane decreased in the U251 and the U138 groups compared with the control group (the U251 treated group:77.8 ±6.4,the control group:124.0 ± 13.4,P =0.000;the U138 treated group:25.8 ±4.2,the control group:54.8 ± 7.1,P =0.000).Conclusions The low expression of IKKε can effectively inhibit the invasion and migration of glioma cells,and the high expression of IKKε can enhance their invasion and migration abilities.Its possible mechanism is to influence the invasion and migration abilities of glioma cells through regulating the expression level of E-cadherin.%目的 探讨胶质瘤细胞中IKKε对E-cadherin表达的调控作用及其对胶质瘤细胞迁移和侵袭能力的影响.方法 构建IKKε过表达载体及IKKε shRNA慢病毒,以IKKε过表达质粒转染TP483胶质瘤细胞株,上调胶质瘤细胞中IKKε的表达水平;以IKKε shRNA慢病毒转染U251、U138胶质瘤细胞株,下调胶质瘤细胞中IKKε的表达水平.采用RT-PCR和Western blot检测细胞中IKKε及E-cadhenn的表达;应用免疫荧光法观察转染后IKKε及E-cadherin表达的变化.通过划痕实验和Transwell法分别检测细胞的迁移和侵袭能力.结果 与未转染组(TP483对照组)相比,转染IKKε的过表达质粒组(TP483处理组)的IKKε蛋白的表达水平升高(P =0.000),而E-cadherin蛋白的表达水平降低(P=0.000).转染IKKε shRNA的胶质瘤细胞(U251处理组和U138处理组)中IKKε蛋白的表达水平较未转染组(U251对照组和U138对照组)明显降低(均P＜0.05),同时各处理组E-cadherin蛋白的表达水平均较其对照组升高(均P ＜0.05).TP483处理组划痕修复率较对照组高[(56.39±0.93)％对比(46.43±1.68)％,P=0.035)].过表达质粒转染24h后,TP483处理组穿过滤膜的细胞数目较对照组增加[(55.8±6.0)个对比(24.6±2.3)个,P =0.000].U251、U138处理组划痕修复率较其各自的对照组降低[U251处理组:(50.40±1.09)％,U251对照组:(87.29±11.11)％,P=0.043;U138处理组:(48.20±1.34)％,U138对照组:(73.15±6.62)％,P=0.035)].IKKε shRNA转染24h后,U251、U138处理组穿过滤膜的细胞数目较对照组少([U251处理组:(77.8±6.4)个,U251对照组:(124.0±13.4)个,P=0.000;U138处理组:(25.8±4.2)个,U138对照组:(54.8±7.1)个,P=0.000].结论 IKKε低表达可有效抑制胶质瘤细胞的侵袭和迁移,而IKKε高表达可增强其侵袭和迁移能力.其可能的机制是通过调节E-cadherin的表达水平从而影响胶质瘤细胞的侵袭和迁移能力.
展开▼
