KLF4调控上皮细胞-间叶细胞转化抑制脑膜瘤

摘要

Objective To study the role of KLF4 in meningioma and its effects on endothelial-mesenchymal transition. Methods Brain arachnoid tissues were collected by surgery and the grade was diagnosed and classified. Western blotting and quantitative real-time PCR methods were used to test protein and mRNA expression of KLF4 in tissue samples. Ectopic KLF4 expression plasmid vector was constructed and gained vi-ral supernatant was used to transfect primary meningioma cell and IOMM-Lee cell,and the chemotactic and invasive activities of cells were meas-ured. At the same time,expressions of E-cadherin,α-catenin,Vimentin and VEGFA in meningioma cell transfected with KLF4 overexpression were detected to study effects of KLF4 overexpression on EMT progress. Luciferase reporter gene assay was utilized to explore effects of KLF4 over-expression on VEGFA promoter,and chromatin immunoprecipitation was performed to test the interaction between them. Results In Vivo,protein levels of KLF4 in meningioma patients were less than that in health group( P ﹤0. 001),grade II was less than grade I( P ﹤0. 05),and grade III was less than grade II( P ﹤0. 05). In Vitro,KLF4 overexpression suppressed chemotaxis and invasion of primary meningioma and IOMM-Lee cells,as the chemotactic and invasive cell number was less than that of control cell groups( P ﹤0. 01). In the experiments of effects of KLF4 overexpression on EMT,E-cadherin andα-catenin associated with endothelial cell showed a higher expression,while Vimentin and VEGFA re-ferred to mesenchymal cell had a less expression compared with control cell groups( P ﹤0. 05). Their protein and mRNA expressions were con-sistent. Results of luciferase reporter gene and ChIP assays have showed that overexpressed KLF4 interacted with VEGFA and suppressed the activ-ity of VEGF promoter. Conclusion KLF4,which is negatively related with the clinical stage of brain arachnoid,can regulate EMT and suppress meningioma.%目的：研究KLF4在脑膜瘤中的作用及其对上皮细胞-间叶细胞转化（ EMT）的调控作用。方法通过临床手术和诊断获得分级脑膜瘤脑蛛网膜组织样本，采用Western blotting和定量real-time PCR对组织样本进行KLF4蛋白半定量表达和mRNA定量表达检测。构建KLF4异位表达载体并获取病毒液对原代脑膜瘤细胞和恶性脑膜瘤细胞系IOMM-Lee进行感染，通过细胞趋化和侵袭实验检测KLF4超表达对脑膜瘤细胞的影响。同时通过检测KLF超表达细胞中EMT相关转录因子（ E-cadherin，α-catenin，Vimentin，VEGFA）的表达探讨其对在该生物学过程中的作用机制。利用荧光素酶报告基因系统测定 KLF4超表达对 VEGFA启动子活性的影响，并进行染色质免疫沉淀实验检测KLF4与VEGFA的相互作用。结果在脑膜瘤患者中，KLF4的蛋白表达低于健康对照组（ P ﹤0.001），且脑膜瘤患病组Ⅰ、Ⅱ和Ⅲ级组间呈梯度下降（ P ﹤0.05）。体外试验中，KLF4超表达细胞组的趋化细胞和侵袭细胞数明显低于对照组（ P ﹤0.01）。KLF4超表达抑制原代脑膜瘤细胞和IOMM-Lee的趋化和侵袭作用。KLF4超表达对EMT的影响实验发现实验组细胞中上皮细胞标记因子E-cadherin和α-catenin表达明显高于对照组（ P ﹤0.05），而间叶细胞标记因子Vimentin和VEGFA的表达显著低于对照组（ P ﹤0.05）。蛋白表达结果与mRNA表达结果相一致。荧光素酶报告基因和染色质荧光免疫实验显示，超表达的KLF4与VEGFA相互作用并抑制VEGFA启动子活性。结论 KLF4蛋白与脑膜瘤患者临床分期有明显的负相关性，且其可以通过调控脑膜瘤细胞上皮细胞-间叶细胞转化而发挥抑制作用。