Rb基因通过MEG3调控β-catenin蛋白磷酸化修饰影响视网膜母细胞瘤侵袭转移的分子机制研究

Inactivation of Rb gene is the main cause. Our previous studies have shown that long non-coding RNA-MEG3 is down-regulation in retinoblastoma due to promoter methylation and is closely related to tumor metastasis. MEG3 could bind to β-Catenin and、GSK-3 βproteins directly too. Other Studies also have shown that Rb protein can regulate the expression of DNA methyltransferase I, and GSK-3β could phosphorylate β-Catenin. Based on this, we speculated that inactivation of Rb gene leads to methylation of MEG3 gene, reduction of degradation of β-Catenin protein, activation of Wnt pathway, and promotion of the invasion and metastasis of retinoblastoma. Here, the methylation regulation of MEG3 promoter by Rb was analyzed by bisulfite sequencing PCR, and the specific binding sites of MEG3 with β-Catenin were analyzed by RNA Pull down and RIP. Role of MEG3 as a "Molecular Scaffold" in Promoting the degradation of β-Catenin was analysed by Phosphorylation of β-Catenin Protein assay and Ubiquitination assay. Effects of Rb-MEG3-β-Catenin on invasion and metastasis was valued in vitro and in vivo. The mechanism of Rb-MEG3-β-Catenin promoting metastasis of retinoblastoma was elucidated to provide a direction for targeted therapy of metastatic retinoblastoma.

Rb基因失活是视网膜母细胞瘤最主要病因。我们前期研究发现LncRNA-MEG3在视网膜母细胞瘤中因启动子甲基化而失活、与肿瘤转移相关，并与β-Catenin、GSK-3β结合。且已知Rb蛋白可调控DNA甲基转移酶Ⅰ表达，GSK-3β可磷酸化β-Catenin。据此我们推测：Rb基因失活导致MEG3基因启动子甲基化失活、β-Catenin磷酸化后泛素化降解减少、Wnt通路激活，导致肿瘤转移。本项目通过重亚硫酸盐测序法分析Rb对MEG3启动子甲基化调控；RNA Pull down、RIP分析MEG3与β-Catenin、GSK-3β结合位点；磷酸化、泛素化分析明确MEG3作为“分子支架”促进β-Catenin磷酸化后泛素化降解；细胞及动物实验分析Rb-MEG3-β-Catenin对侵袭能力影响；最终阐明Rb-MEG3-β-Catenin促进肿瘤转移机制，为转移性视网膜母细胞瘤靶向治疗提供方向。

在我们之前的研究中，我们发现LncRNAMEG3的低表达与视网膜母细胞瘤的侵袭转移密切相关，但是MEG3失活诱导视网膜母细胞瘤细胞系侵袭和转移的分子机制尚不清楚。我们使用GEO数据库分析了MEG3在视网膜母细胞瘤组织中MEG3的表达和MEG3相关通路。采用划痕、跨孔迁移、小鼠肿瘤转移和小鼠荧光活体成像实验检测视网膜母细胞瘤细胞系的迁移和侵袭。通过RNA pull down、电泳迁移率转移、RIP、免疫共沉淀和泛素化分析来分析其分子机制。GEO数据库显示，MEG3在视网膜母细胞瘤组织中表达较低，与视网膜母细胞瘤细胞的侵袭和Wnt通路的活性密切相关。体内和体外实验均证实，MEG3可抑制视网膜母细胞瘤细胞的迁移和侵袭。细胞实验证实，MEG3可间接促进β-catenin与GSK-3β的结合，并间接诱导β-catenin的磷酸化、泛素化和降解。综上所述，MEG3可通过GSK-3β促进β-catenin的降解，从而使Wnt通路失活，最终抑制视网膜母细胞瘤细胞的侵袭和转移，为转移性视网膜母细胞瘤靶向治疗提供方向。.。

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