摘要
【目的】研究过表达整合素链接激酶(ILK)对胶质瘤细胞上皮间质转化(EMT)的调控作用及初步机制。【方法】项目组前期实验已经成功构建了重组质粒p EGFP-C1-ILK,并将其转染给SHG-44胶质瘤细胞,通过G418筛选出了稳定转染的细胞株。实验分组如下:SHG-44(空白对照组)、p EGFP-C1(空载组),及p EGFP-C1-ILK(稳转组),先检测各组中ILK的表达情况(RNA及蛋白质水平)及侵袭能力,再检测过表达ILK后对EMT标记物的影响,最后再分别应用NF-κB通路的特异性阻断剂BAY11-7028和RNA沉默的方法阻断核因子NF-κB通路,Western blot方法检测上皮间质转化标记物Ecadherin在阻断前及阻断后的表达情况,初步探讨NF-k B通路在过表达ILK的胶质瘤细胞中对EMT的调控作用。【结果】稳转组中ILK明显过表达,同时侵袭能力增强(Transwell小室透膜细胞数在对照组、空载组和稳转组分别为:92,87,229)。Western blot检测EMT标记物蛋白的表达:稳定转染组中snail,slug,twist,vimentin的表达较对照组及空载组的表达明显增高,而E-cadherin的表达则在稳定转染组中明显降低,差异有统计学意义(P<0.05)。当分别用NF-k B特异性阻断剂BAY11-7028及p65 si RNA的方法阻断该通路后,稳定转染组中E-cadherin蛋白表达明显升高。【结论】胶质瘤中过表达ILK可使侵袭能力增强,同时可下调E-cadherin,上调vimentin及Snail,Slug,Twist的表达,可能通过此机制促进脑胶质瘤细胞的上皮间质转化,NF-k B通路可能参与、调控该进程。
[Objective] To research the effect and preliminary mechanism of integrin linked kinase （ILK）on epithelial mesenchymal transition （EMT） in human glioma cells.[Methods] The recombinant plasmid pEGFP - C 1-ILK was successfhlly constructed and transfected into SHG-44 glioma cells and the stable transfected cell lines were selected by G418 previously by our project team. The experimental group is as follows： SHG-44 （mock group）,pEGFP-C1 （empty vector group）,and pEGFP-CI-ILK （stable transition group）. The expression of ILK and invasive ability were detected in every group at first. Then, the effect of overexpression of ILK on the EMT markers was tested. At last, by using the method of RNA silence and specific blockers BAY11- 7028 separately, the NF-kB pathway was blocked. The expression of E-cadherin was detected by western blot to determine the effect of NF-kB pathway on the regulation of EMT in ILK-overexpressed cells. [ Results ] Overexpression of ILK, meanwhile, invasive ability enhancement（the result of transwell： number of cell migration in mock, empty vector and stable transfection group is 92, 87, 229） were found in the stable transfection group. Expression of EMT marker-protein was detected by western blot, and the expression of snail, slug, vimentin, twist in the stable transfection group was significantly higher than that in the control group and the empty vector group, while the expression of E-cadherin was significantly decreased in the stable transfection group, and the difference was statistically significant （P 〈 0.05）. E-cadherin protein expression was significantly increased in the stable transfection group after BAY11-7028 and P65 SiRNA were used to block the pathway of NF-kB respectively. [Conclusion ] Overexpression of ILK can make invasive ability enhanced, and down regulate E-cadherin, up regulate the expression of vimentin and Snail, Slug and Twist, which may promote the epithelial mesenchymal transition of glioma cells, and NF-kB pathway may participate in the process of regulation.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2016年第2期210-216,共7页
Journal of Sun Yat-Sen University:Medical Sciences
基金
辽宁省博士启动基金(201501101)
