WIF-1基因启动子甲基化对星形胶质瘤生物学行为的影响
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摘要
背景和目的Wnt/p-catenin信号通路参与了包括星形胶质瘤在内的多种肿瘤的发生发展过程。WIF-1是Wnt/β-catenin信号通路的拮抗因子,有研究发现由于其启动子的甲基化导致WIF-1表达减少甚至缺失,Wnt信号通路异常活化,从而引起肿瘤的发生发展。但是,上述变化机制在星形胶质瘤中尚未见报道。因此,本研究拟分析星形胶质瘤中WIF-1的表达情况和启动子甲基化状态,以及WIF-1启动子甲基化对星形胶质瘤细胞的生物学行为影响,从而确定启动子甲基化是否是星形胶质瘤中WIF-1表达沉默的主要机制,并证明WIF-1在星形胶质瘤的发生发展中起重要作用。
研究方法首先,用免疫组化、半定量的RT-PCR和甲基化特异性PCR检测了35例星形胶质瘤和4例正常脑组织中WIF-1的表达情况和启动子甲基化状态,分析了WIF-1的表达和启动子甲基化状态的关系；然后,用去甲基化药物5-氮-2'-脱氧胞苷处理胶质母细胞瘤来源的U251细胞系和3例原代肿瘤细胞,分析药物作用前后WIF-1的表达和启动子甲基化状态；最后,我们将WIF-1的表达载体稳定转染U251细胞系,通过细胞增殖实验、克隆形成实验和流式细胞术(FCM)分析WIF-1基因对U251细胞系生物学行为的影响。
结果与正常脑组织相比,星形胶质瘤中WIF-1 mRNA和蛋白的表达明显降低。35例星形胶质瘤中,20例(57.14%)发现WIF-1基因启动子甲基化。统计分析发现WIF-1基因启动子甲基化与其表达下降明显相关。U251细胞系和3例原代肿瘤细胞被去甲基化药物5-氮-2'-脱氧胞苷处理后,WIF-1基因表达恢复。将WIF-1的表达载体转染U251细胞系后,细胞的生长和克隆形成明显受抑制,G0/G1期细胞增多,S期和G2/M期细胞减少。
结论在星形胶质瘤中,WIF-1基因启动子甲基化是其表达下降的重要原因,并且导致了肿瘤细胞生物学行为的改变。WIF-1基因启动子甲基化可能是星形胶质瘤发生发展的重要原因。
Background:Wnt/β-catenin pathway may play a major role in the tumorigenesis and tumor progression in many tumor types including astrocytomas.Wnt inhibitory factor-1(WIF-1) acts as a Wnt-antagonists and tumor suppressor, but hypermethylation of WIF-1 gene promoter and low expression activate Wnt signaling aberrantly and induce the development of various human tumors. With this work we intended to investigate the relation between expression and promoter methylation status of WIF-1 gene in human astrocytomas, and illuminate the impact of WIF-1 gene in astrocytomas with WIF-1 silencing.
Methods:Firstly,we analysed expression and methylation status of WIF-1 in 35 astrocytomas and 4 normal brain tissues by immunohistochemistry, semiquantitative RT-PCR and methylation specific PCR. The relationship between methylation and expression of the genes was analyzed. Secondly, U251 cell lines and primary-culture cells derived from 3 patients with glioblastoma were treated with demethylating agent,5-aza-2'-deoxycytidine (5-aza-CdR), to further examine the role of methylation in silencing of WIF-1. Finally, we restored the expression of WIF-1 by transfer with a pReceiver-M03-WIF1 vector into U251 cell lines lacking WIF-1 expression and analysed the effects of WIF-1 on U251 cell lines by cell proliferation, colony formation, and Flowcytometry(FCM).
Results:Significant downregulation of WIF-1 mRNA and protein expression was observed in astrocytoma tissues compared with normal brain tissues. WIF-1 gene aberrant methylation was observed in 20(57.14%) of 35 tumor samples. Hypermethylation in the WIF-1 promoter region correlated with downregulation of WIF-1 expression in astrocytoma tissues. Moreover, treatment with demethylating 5-aza-2'-deoxycytidine restored WIF-1 expression in U251 cell lines and primary-culture cells derived from 3 patients with glioblastoma. Transfection of the WIF-1 gene into U251 cell lines resulted in a significant inhibition on cell proliferation and colony formation and an increased fraction of cells in the G0/G1-phase and subsequent decrease in S-phase and G2/M-phase.
Conclusion:Our results demonstrate that the WIF-1 gene is frequently down-regulated or silenced in astrocytomas by aberrant promoter methylation and that this epigenetic alteration is involved in glioma cell proliferation and cell cycle. This may be an important mechanism in astrocytoma carcinogenesis.
研究方法首先,用免疫组化、半定量的RT-PCR和甲基化特异性PCR检测了35例星形胶质瘤和4例正常脑组织中WIF-1的表达情况和启动子甲基化状态,分析了WIF-1的表达和启动子甲基化状态的关系；然后,用去甲基化药物5-氮-2'-脱氧胞苷处理胶质母细胞瘤来源的U251细胞系和3例原代肿瘤细胞,分析药物作用前后WIF-1的表达和启动子甲基化状态；最后,我们将WIF-1的表达载体稳定转染U251细胞系,通过细胞增殖实验、克隆形成实验和流式细胞术(FCM)分析WIF-1基因对U251细胞系生物学行为的影响。
结果与正常脑组织相比,星形胶质瘤中WIF-1 mRNA和蛋白的表达明显降低。35例星形胶质瘤中,20例(57.14%)发现WIF-1基因启动子甲基化。统计分析发现WIF-1基因启动子甲基化与其表达下降明显相关。U251细胞系和3例原代肿瘤细胞被去甲基化药物5-氮-2'-脱氧胞苷处理后,WIF-1基因表达恢复。将WIF-1的表达载体转染U251细胞系后,细胞的生长和克隆形成明显受抑制,G0/G1期细胞增多,S期和G2/M期细胞减少。
结论在星形胶质瘤中,WIF-1基因启动子甲基化是其表达下降的重要原因,并且导致了肿瘤细胞生物学行为的改变。WIF-1基因启动子甲基化可能是星形胶质瘤发生发展的重要原因。
Background:Wnt/β-catenin pathway may play a major role in the tumorigenesis and tumor progression in many tumor types including astrocytomas.Wnt inhibitory factor-1(WIF-1) acts as a Wnt-antagonists and tumor suppressor, but hypermethylation of WIF-1 gene promoter and low expression activate Wnt signaling aberrantly and induce the development of various human tumors. With this work we intended to investigate the relation between expression and promoter methylation status of WIF-1 gene in human astrocytomas, and illuminate the impact of WIF-1 gene in astrocytomas with WIF-1 silencing.
Methods:Firstly,we analysed expression and methylation status of WIF-1 in 35 astrocytomas and 4 normal brain tissues by immunohistochemistry, semiquantitative RT-PCR and methylation specific PCR. The relationship between methylation and expression of the genes was analyzed. Secondly, U251 cell lines and primary-culture cells derived from 3 patients with glioblastoma were treated with demethylating agent,5-aza-2'-deoxycytidine (5-aza-CdR), to further examine the role of methylation in silencing of WIF-1. Finally, we restored the expression of WIF-1 by transfer with a pReceiver-M03-WIF1 vector into U251 cell lines lacking WIF-1 expression and analysed the effects of WIF-1 on U251 cell lines by cell proliferation, colony formation, and Flowcytometry(FCM).
Results:Significant downregulation of WIF-1 mRNA and protein expression was observed in astrocytoma tissues compared with normal brain tissues. WIF-1 gene aberrant methylation was observed in 20(57.14%) of 35 tumor samples. Hypermethylation in the WIF-1 promoter region correlated with downregulation of WIF-1 expression in astrocytoma tissues. Moreover, treatment with demethylating 5-aza-2'-deoxycytidine restored WIF-1 expression in U251 cell lines and primary-culture cells derived from 3 patients with glioblastoma. Transfection of the WIF-1 gene into U251 cell lines resulted in a significant inhibition on cell proliferation and colony formation and an increased fraction of cells in the G0/G1-phase and subsequent decrease in S-phase and G2/M-phase.
Conclusion:Our results demonstrate that the WIF-1 gene is frequently down-regulated or silenced in astrocytomas by aberrant promoter methylation and that this epigenetic alteration is involved in glioma cell proliferation and cell cycle. This may be an important mechanism in astrocytoma carcinogenesis.
引文
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