EZH2/linc00261/FOXA2轴调控肝细胞癌转移复发及其机制研究

Epithelial-mesenchymal transition (EMT) and tumor stem cells (CSCs) are important causes of hepatocellular carcinoma (HCC) metastasis and recurrence. Previous studies found that the low expression of linc00261 is an independent risk factor for poor prognosis after HCC radical resection. Its expression, which is closely related to its methylation level of CpG islands in promoter and H3K27Me3 expression, is inhibited by EZH2. Meanwhile, linc00261 triggers the transcription and expression of its adjacent gene FOXA2, subsequently affecting the EMT process and CSCs properties in liver cancer cells. Therefore, we have a hypothesis that linc00261 may be epigenetically silenced by EZH2, and result in the occurrence of EMT and the acquirement of CSCs traits by regulating the transcription and expression of FOXA2, and lead to the metastasis and recurrence of HCC. This study intends to clarify that the molecular mechanism of EZH2 silencing linc00261, the transcriptional regulation of FOXA2 by linc00261, followed by the regulation of EMT, the acquirement of CSCs traits and the involved signaling pathways modulated by EZH2/linc00261/FOXA2 axis using molecular biotechnology, cell and animal models, series of rescue experiments and clinical specimen detection. The work will discover a new prognostic biomarker after HCC radical resection and the candidates of therapeutic targets for anti-metastasis and recurrence.

上皮间质转化（EMT）和肿瘤干细胞（CSCs）是导致肝细胞癌（HCC）转移复发的重要原因。前期研究发现：linc00261的低表达是HCC术后预后不良的独立危险因素；而其表达下调与EZH2介导的启动子CpG岛甲基化和H3K27Me3密切相关，linc00261激活邻位基因FOXA2的转录和表达，影响HCC的EMT和CSCs特性。因此，EZH2可能表观沉默linc00261，进而调控FOXA2的转录与表达，促进HCC发生EMT及获得CSCs特性，最终导致HCC的转移复发。本项目拟采用分子生物学技术，通过细胞与动物模型、系列补救实验，结合临床标本检测，阐明：EZH2沉默linc00261的分子机制；linc00261对FOXA2的转录调控机制；EZH2/linc00261/FOXA2轴对HCC EMT和CSCs特性的调控及其信号途径，以期发现新的HCC预后评估指标和抗转移复发的治疗靶点。

肝细胞癌(HCC)是世界上最常见的恶性肿瘤之一。近几十年来，长链非编码RNA (lncRNAs)受到越来越多的关注，并被报道在人类癌症中发挥重要作用，使其成为疾病评估和治疗的理想候选。在本研究中，我们发现linc00261缺失与HCC恶性生物学行为尤其是MVI有显著相关性，是无复发生存的良好独立预后因素，同时，深入研究证明linc00261在肝癌中抑制上皮间质转化从而抑制细胞的迁移、侵袭和肺转移性病变的形成。此外，linc00261与其相邻基因FOXA2在HCC中呈正相关，功能增益和功能缺失分析表明linc00261在转录上促进了FOXA2的表达。同时，生物信息学分析和拯救试验证实，linc00261通过上调FOXA2表达部分抑制迁移、侵袭和EMT。分子机制研究表明，linc00261通过招募SMAD3，顺式转录上调FOXA2；而EZH2通过调节H3K27在Lys27位点的三甲基化(H3K27Me3)负责linc00261的转录抑制，EZH2和H3K27Me3均与HCC中linc00261的表达呈负相关。.此外，我们发现linc00261在TGF-β1刺激的细胞中下调，并在HCC细胞中过表达linc00261后可减弱EMT和肿瘤干细胞特性。Linc00261还能抑制体外肿瘤的成球能力，降低体内致瘤性。进一步研究发现，linc00261抑制SMAD3的表达和磷酸化，这可能是TGF-β1信号通路介导的EMT和获得肿瘤干细胞特性核心环节。此外，在HCC样本中，linc00261与p-SMAD3呈负相关。.综上所述，我们的研究得出两个重要结论，即：1.表观遗传沉默的linc00261通过诱导FOXA2转录缺陷促进肝细胞癌的转移；2. TGF-β1诱导的linc00261缺失通过调节SMAD3促进肝细胞癌的上皮-间质转化和干性。