LncRNA-NEF转录激活FOXA2并通过抑制β-catenin信号通路抑制肝癌细胞的EMT过程

LncRNA-NEF transcriptionally activates FOXA2 and inhibits epithelial–mesenchymal transition of hepatocellular carcinoma cells by inhibitingβ-catenin signaling pathway

目的探讨长链非编码lncRNA-NEF对肝癌转移关键步骤EMT过程的分子调节机制。方法通过TGF-β诱导肝癌细胞系Hep3B细胞向EMT转化,通过构建pcDNA3.1-FOXA2过表达载体,过表达FOXA2诱导肝癌细胞系HepG2细胞向MET转化,随后检测两种细胞的E-cadherin、Vimentin、Snail共3种EMT相关标志物的表达变化、lncRNA-NEF的表达变化,以及细胞侵袭能力变化。之后检测上述两种细胞分别经历EMT和MET转化后,β-catenin信号通路相关蛋白因子的表达变化情况。结果lncRNA-NEF在肝癌组织和肝癌细胞中表达水平低于对照组正常细胞(P<0.05);与对照组相比,TGF-β处理组的细胞侵袭能力显著增强(P<0.05),pcDNA3.1-FOXA2转染组的细胞侵袭能力显著降低(P<0.05);与对照组相比,TGF-β处理组的Hep3B细胞内磷酸化的β-catenin(p-β-catenin)表达量显著增高,而总β-catenin蛋白水平不变,pcDNA3.1-FOXA2转染组的HepG2细胞内磷酸化的β-catenin(p-β-catenin)表达量显著降低,而总β-catenin蛋白水平不变。LncRNA-NEF是以顺式作用方式转录激活FOXA2,并通过抑制β-catenin信号通路抑制肝癌细胞的EMT过程。结论LncRNA-NEF能转录激活FOXA2并通过抑制β-catenin信号通路抑制肝癌细胞的EMT过程。

Objective To explore the molecular mechanism of long-chain non-coding RNA-neighboring enhancer of FOXA2(lncRNA-NEF)on the epithelial-mesenchymal transition(EMT)process of hepatocellular carcinoma(HCC),a key step in liver cancer metastasis.Methods HCC line Hep3B was treated with transforming growth factor-β(TGF-β)to induce EMT.A pcDNA3.1-FOXA2 overexpression vector was constructed and transduced into another HCC line HepG2 to overexpress FOXA2 and induce mesenchymal-epithelial transition(MET).The EMT-related markers including E-cadherin,Vimentin and Snail were detected and cell invasion assay was performed in these lines with and without relative treatment.The expression of lncRNA-NEF was determined in 10 paired operated liver cancer tissues and adjacent normal tissues,as well as in the HCC lines.Subsequently,the protein expression of factors related toβ-catenin signaling pathway before and after EMT or MET transformation in the HCC lines were detected.Results The expression level of lncRNA-NEF in liver cancer tissues and HCC lines was lower than that in normal controls(P<0.05).Compared with the control cells,the cell invasion ability of TGF-βtreated HCC cells significantly enhanced(P<0.05),but the cell invasion ability of PCDNA3.1-FOXA2 transduced cells significantly reduced(P<0.05).The expression level of phosphorylatedβ-catenin(p-β-catenin)in Hep3B cells treated with TGF-βincreased significantly when compared with the control cells.On the contrary,the expression level of phosphorylatedβ-catenin(P-β-catenin)in HepG2 cells transfected with PCDNA3.1-FoxA2 significantly decreased.The level of totalβ-catenin protein remained unchanged in both HCC lines with and without relative treatment.LncRNA-NEF activated FOXA2 in a cis-acting manner,and inhibited the EMT process of liver cancer cells by inhibiting theβ-catenin signaling pathway.Conclusion LncRNA-NEF activates FOXA2 transcription and inhibits the EMT process of liver cancer cells by inhibiting theβ-catenin signaling pathway.