过表达整合素链接激酶通过NF-kB通路促进脑胶质瘤细胞的上皮间质转化

发布时间：2018-03-13 03:14

本文选题：稳定过表达　切入点：NF-k　出处：《中山大学学报(医学科学版)》2016年02期 　论文类型：期刊论文

【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(P0.05)。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。
[Abstract]:[objective] to study the regulation and mechanism of overexpression of integrin linked kinase (ILK) on epithelial interstitial transformation of glioma cells. [methods] Recombinant plasmid pEGFP-C1-ILK has been successfully constructed and transfected into SHG-44 glioma cells. The stable transfected cell lines were selected by G418. The experimental groups were as follows: SHG-44 (blank control group, pEGFP-C1-ILK) and invasion ability. The effects of ILK expression on EMT markers were also detected. Finally, BAY11-7028 and RNA silencing of NF- 魏 B pathway were used to detect the expression of Ecadherin before and after blocking NF-魏 B pathway by Western blot. To investigate the role of NF-k B pathway in the regulation of EMT in glioma cells overexpression of ILK. [results] the expression of ILK was significantly overexpressed in the stable transfer group, and the invasiveness of ILK was enhanced in the control group, and the number of transwell permeability cells was increased in the control group. The expression of EMT marker protein in the stable transfection group was significantly higher than that in the control group and the no-load group, but the expression of E-cadherin in the stable transfection group was significantly lower than that in the stable transfection group, and the expression of E-cadherin in the stable transfection group was significantly lower than that in the control group and the no-load group, and the expression of E-cadherin in the stable transfection group was significantly lower than that in the control group. The expression of E-cadherin protein increased significantly in stable transfection group after blocking the pathway with NF-k B specific blocker BAY11-7028 and p65si RNA respectively. [conclusion] overexpression of ILK in glioma can enhance the invasiveness. At the same time, E-cadherin could be down-regulated and the expression of vimentin and Sluger-Twist could be upregulated, which may promote the epithelial interstitial transformation of glioma cells and the NF-kB pathway may be involved in the regulation of this process.
【作者单位】：辽宁医学院附属第一医院神经外科;辽宁医学院附属第一医院儿科;
【基金】：辽宁省博士启动基金(201501101)
【分类号】：R739.41

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