Lgr5促进肝癌细胞上皮间质转化的分子机制

The depth of tumor invasion, metastasis, and postoperative relapse are universal and continues to be the main cause of late deaths in patients with liver cancer.Epithelial - mesenchymal transition (EMT) is closely associated with frequent postoperative recurrence and poor survival in patients with hepatocellular carcinoma. The most recent research indicates that EMT is currently a hot topic of among liver cancer researchers. Signaling pathways have been implicated in EMT regulating important processes during the development of hepatocellular carcinoma including Wnt/ -catenin signaling pathway. G- protein coupled receptor 5 (Lgr5),as an important target gene of Wnt signal pathway,is playing an important role in metabolic regulation and signal transduction processes within cells. Our previous work suggested that Lgr5 could promote EMT and enhance liver cancer recurrence involving Wnt/β -catenin signal transduction pathway.However, the molecular mechanism underlying of Lgr5-induced EMT was not clear. Here, we will explore the possible epigenetic mechanisms of Lgr5 gene aberrant expression in liver cancer and screen invasion/metastasis-associated gene interacts with LGR5 to stimulate tumor recurrence on a genome-wide scale. Then we will focus on the the molecular mechanism of how LGR5 regulate the expression of β-catenin. At last,we will identify the interaction domains of Lgr5 and targeted protein by using two-hybrid yeast technique. In a word, we want to know whether Lgr5 is playing a key role or not in invasion and metastasis of hepatocellular carcinoma and provide a new target site for the treatment of liver cancer.

肝癌的侵袭转移和术后复发是肝癌患者死亡的主要原因。上皮-间质转化(EMT)与肝癌复发及转移密切相关, 已成为当前生命科学研究的热点。多种信号转导通路参与了肿瘤细胞的EMT过程,其中Wnt/β-catenin信号通路尤为引起人们的关注。作为Wnt 信号通路的重要靶基因，G-蛋白偶联受体5(Lgr5)，在体内细胞信号转导和代谢调节等方面发挥着重要作用。我们前期工作提示，LGR5可能是通过Wnt/β-catenin信号传导通路介导原发性肝癌的上皮间质转化。为探求其具体分子机制，我们将从表观遗传的角度探讨研究Lgr5基因的表达调控机制；运用蛋白组学技术筛选与Lgr5相关的侵袭转移蛋白；并将重点研究Lgr5对β-catenin的表达调控机制；最后采用酵母双杂交鉴定Lgr5与靶蛋白相互作用结构域。以其明确Lgr5在肝癌细胞侵袭转移过程中是否起关键作用，为肝癌治疗提供新的靶位点。

上皮–间质转化(EMT)与肝肿瘤复发及转移密切相关,多种信号转导通路参与了肝肿瘤细胞的EMT过程,其中Wnt/β-catenin信号通路发挥重要作用。作为Wnt 信号通路的重要靶基因，富含亮氨酸重复序列G-蛋白偶联受体5(Lgr5)功能涉及体内细胞信号转导和代谢调节等方面。本课题发现Lgr5的高表達與腫瘤大小、 腫瘤數量、巴塞罗那分期以及門靜脈癌栓呈正相關。多因素分析提示Lgr5的高表達是肝癌患者預後不良的獨立危險因素。肝癌细胞的侵袭转移过程中存在明显的EMT现象。Lgr5发挥促癌作用与原发性肝癌侵袭转移过程中EMT有关，机制是通过Wnt/β-catenin信号传导通路介导原发性肝癌EMT的发生。进一步分析显示Lgr5促进了肝癌细胞对阿霉素的耐药性。PDCD5作为Lgr5的靶分子在介导阿霉素耐藥過程中發揮作用，過表達PDCD5可以明显提高肝癌細胞對阿霉素的敏感性。PDCD5能抑制Lgr5介导的對阿霉素的耐藥。PDCD5以及敲除Lgr5后克隆形成实验，验证PDCD5對提高肝癌細胞阿霉素敏感性的作用。机制研究提示，Lgr5可以通过结合PDCD5以阻止其入核来影响p53的稳定性并促进其降解。更重要的是，Lgr5可以抑制阿霉素介导的通过p53途径的凋亡以及降低对阿霉素的敏感性。另外，我们发现长链非编码RNA：SNHG6可以发挥促肝癌生长作用。机制上，SNHG6可以作为ceRNA通过竞争性结合miR-101-3p上调ZEB1的表达，诱导EMT的发生，促进HCC的侵袭转移。SNHG6可以作为抗HCC侵袭、转移治疗的潜在靶点。本课题在肝癌组织小RNA测序中发现一个新的miRNA,来源于假基因MTCO3P38，暂命名为miR-MTCO3P38，长度为18nt,经过RNA二级结构预测、PCR产物二代测序与northern blot等试验验证其的确稳定表达于肝组织和肝细胞系中。功能上miR-MTCO3P38主要抑制肝癌细胞的侵袭迁移行为。机制方面，miR-MTCO3P38主要通过靶基因TMOD1发挥作用。