WNT4在脑胶质瘤中甲基化下调及抑制恶性胶质瘤细胞增殖的研究

WNT4 down-regulates methylation of glioma and inhibits malignant glioma cell proliferation

目的探讨WNT4在脑胶质瘤细胞系和组织中细胞系的表达与甲基化对脑胶质瘤细胞系U87增殖的作用和机制。方法qRT-PCR和免疫蛋白印迹实验分析WNT4在脑胶质瘤组织中的表达和甲基化及WNT4在脑胶质瘤细胞系中的表达;以胶质瘤U87细胞为本实验研究对象,分别转染慢病毒载体pLVX-Gfp-WNT4(实验组)和pLVX-Gfp-NC(对照组);细胞增殖和克隆形成实验分析WNT4基因对细胞增殖的影响;免疫蛋白印迹法验证WNT4对Wnt/β-catenin信号通路的影响。结果与正常人星形胶质细胞相比,WNT4 mRNA在脑胶质瘤细胞系中的表达下调(P<0.001),与正常脑组织相比,WNT4 mRNA和蛋白在脑胶质瘤组织中表达下调(P<0.001);脑胶质瘤分级越高WNT4 mRNA表达越低(P<0.001),高表达WNT4 mRNA患者的预后好(P<0.001);WNT4在脑胶质瘤中甲基化程度明显高于正常脑组织。过表达WNT4后细胞活性和增殖能力受到抑制(P<0.001);恢复WNT4表达后Wnt/β-catenin信号通路及其下游细胞因子明显受到抑制。结论WNT4在人脑胶质瘤组织中表达甲基化下调,通过抑制Wnt/β-catenin信号通路参与脑胶质瘤的进展。

Objective The aims of this study were to investigate the expression and methylation of WNT4 in glioma tissues and malignant glioma cell lines,and its effect and mechanism on the proliferation of glioma U87 cell line.Methods qRT-PCR and Western blot were used to analyze the expression and methylation of WNT4 in human glioma tissues and the expression of WNT4 in U87 cells.MSP was used to analysis the methylation of Wnt4 in glioma tissues.U87 cells were used as experimental cells transfected with lentiviral vectors pLVX-Gfp-WNT4(experimental group)and pLVX-Gfp-NC(control group).Cell proliferation and clone formation experiments were performed to analyze the effect of WNT4A gene on cell proliferation.Western blot was used to verify the effect of WNT4 on the WNT/β-catenin signal pathway.Results Compared with normal human astrocytes,WNT4 mRNA and protein were significantly down-regulated in glioma tissues and cells(P<0.001).WNT4 methylation was significantly increased in gliomas tissues.The expression of WNT4 was related to the grade of glioma,and the good prognosis of patients with the high expression of WNT4 mRNA;the degree of methylation of WNT4 in glioma tissues was significantly higher than that of normal brain tissues.The Re-expression of WNT4 in significantly decreased in cell viability and proliferation of U87 cells(P<0.001);The WNT/β-catenin signaling pathway and its downstream cytokines were significantly suppressed after WNT4 expression was restored in U87 cells.Conclusion The expression of WNT4 is down-regulated in human glioma tissues and participates in the progression of gliomas by inhibiting the Wnt/β-catenin signaling pathway.