肝细胞癌中转录因子E2F7对Wnt/β-catenin信号通路活性的影响

The Effect of Transcription Factor E2F7 on Wnt/β-catenin Signaling Pathway in Hepatocellular Carcinoma

探讨肝细胞癌(HCC)中非典型性E2F家族成员E2F7在肝癌细胞生长、分化中的作用及可能涉及的分子机制.本研究运用实时荧光定量PCR检测38例原发性肝细胞癌及对应的癌旁组织中E2F7基因mRNA的表达情况;分别通过基因过表达和RNA干扰技术上调或下调E2F7基因表达,并运用实时荧光定量PCR和Western印迹检测肝癌细胞株MHCC-H中β-catenin及其靶基因cmyc的表达情况;双荧光素酶报告基因系统检测E2F7对Wnt/β-catenin信号通路活性的影响;核浆分离实验检测过表达E2F7基因对β-catenin入核的影响;免疫共沉淀实验检测异位表达E2F7与内源β-catenin的相互作用.结果显示,肝细胞癌组织中E2F7基因的表达量显著高于相应的癌旁组织(P<0.001);转录因子E2F7可与β-catenin相互作用并促进β-catenin进入细胞核.转录因子E2F7可以促进Wnt/β-catenin信号通路的活性.

To explore role of transcription factor E2F7 on Wnt/β-catenin signaling pathway in hepatocellular carcinoma （HCC） cells, the expression of E2F7 mRNA was analyzed using realtime PCR in 38 pairs of primary HCC and corresponding adjacent tissues. In MHCCH cells, over expression and RNA interference technology were applied to up-regulate or downregulate the E2F7 gene expression. Changes of β-catenin and downstream c-myc gene expression were detected by real-time PCR and immunoblot assays. Furthermore, dual-luciferase reporter assay was used to evaluate the effect of E2F7 on Wnt/β-catenin signal pathway. Nuclear and cytoplasmic separation of β-catenin was performed to measured the over expression effect of E2F7 on its activation in the nucleus.T he interaction between exogenous E2F7 and endogenous β-catenin was verified by coimmunoprecipitation. The results suggested that the mRNA expression level of E2F7 in HCC tissues was significantly higher than adjacent tissues （P 〈0.001）. E2F7 interacted with βcatenin increased the translocation of β-catenin into the nucleus, thus to increase the activity of Wnt/β-catenin signaling pathway.