DNA双加氧酶TET家族蛋白1抑制上皮性卵巢癌转移的作用机制研究

Research on the mechanism of action of DNA dioxygenase TET family protein 1 inhibiting the metastasis of epithelial ovarian cancer

目的探讨TET家族蛋白1(TET1)在调控上皮性卵巢癌(EOC)转移中的作用机制。方法在EOC细胞系中瞬时转染TET1过表达质粒,通过Transwell实验检测EOC细胞迁移能力变化。通过蛋白质免疫印迹检测上皮细胞黏附分子(EpCAM)的表达。通过蛋白质免疫荧光实验检测EpCAM上游转录因子β-连环蛋白的亚细胞定位变化。检测β-连环蛋白的上游抑制因子叉头框基因O4(FOXO4)基因启动子区5hmC和5mC含量变化。通过染色质免疫共沉淀实验检测TET1与FOXO4基因启动子区域结合情况。利用EOC组织芯片对TET1与β-连环蛋白进行免疫组织化学染色,探讨二者表达的相关性。结果过表达TET1明显抑制EOC细胞系OVSAHO和JHOS4的细胞迁移能力,且明显抑制EpCAM的表达。TET1过表达后促进β-连环蛋白的亚细胞定位发生变化,其核内聚集明显减少,β-连环蛋白与EpCAM基因启动子区域结合亦明显减少。TET1过表达可以促进FOXO4的表达,进而抑制β-连环蛋白进入细胞核发挥转录因子作用。当FOXO4表达被敲低后,β-连环蛋白的细胞核内分布明显增加。在浆液性卵巢腺癌组织样本中,71.8%(28/39)TET1表达呈阳性,15.4%(6/39)β-连环蛋白表达呈阳性,TET1与β-连环蛋白的表达呈负相关(χ^(2)=4.745,P=0.030)。结论TET1通过调控FOXO4/β-连环蛋白/EpCAM轴抑制EOC转移。

Objective To investigate the mechanism of TET family protein 1(TET1)in regulating epithelial ovarian cancer(EOC)metastasis.Methods TET1 overexpression plasmid was transiently transfected into EOC cell lines,and cell migration was detected by Transwell assay.The expression of epithelial cell adhesion molecules(EpCAM)was detected by western blot,and the subcellular localization ofβ-catenin,the upstream transcription factor of EpCAM,was detected by immunofluorescence staining.The contents of 5hmC and 5mC in the promoter region of Forkhead box subclass O protein 4(FOXO4),the upstream inhibitor ofβ-catenin,were detected.The binding of TET1 protein with FOXO4 gene promoter region was detected by chromatin immunocoprecipitation test.Immunohistochemical staining was performed to investigate the correlation between TET1 andβ-catenin expression.Results Overexpression of TET1 significantly inhibited cell migration of EOC cell lines OVSAHO and JHOS4,and significantly inhibited the expression of EpCAM.Mechanistically,overexpression of TET1 altered the subcellular localization ofβ-catenin,which is upstream transcription factor of EpCAM,and its nuclear aggregation significantly decreased.The binding betweenβ-catenin and EpCAM gene promoter region was also significantly reduced.Overexpression of TET1 promoted the expression of FOXO4,which is upstream inhibitory factor ofβ-catenin.When FOXO4 expression was knocked down,the distribution ofβ-catenin in the nucleus was significantly increased.In the tissue samples of serous ovarian adenocarcinoma,71.8%(28/39)had TET1 positive expression,15.4%(6/39)hadβ-catenin positive expression,and the expression of TET1 was negatively correlated withβ-catenin(χ^(2)=4.745,P=0.030).Conclusion TET1 inhibits the metastasis of epithelial ovarian cancer by regulating FOXO4-β-catenin-EpCAM axis.