LncRNA ETAPOD1作为miR-101b-3p的海绵调控肝细胞癌的侵袭迁移能力

LncRNAs plays an important role in the invasion and metastasis of hepatocarcinoma. However, the specific mechanism has not been completely clarified. The applicant found that the down-regulation of a new type of LncRNA ETAPOD1(EMT and invadopodia-related LncRNA 1) and Dlg5 after knockdown oncogene Hint1, and Dlg5 could inhibit the formation of invasive pseudopodia and the epithelial-mesenchymal transformation of Hepatocellular carcinoma cells. And the knockdown of ETAPOD1 induced the down-regulation of Dlg5, the formation of invasive pseudopodia and the epithelial-mesenchymal transformation. Biological information screening and luciferase experiments confirmed that miR-101b-3p targeted regulation of Dlg5. The project intends to study whether ETAPOD1 competitively binds miR-101b-3p through the ceRNA mechanism to up-regulate Dlg5 to inhibit the formation of invasive pseudopodia and epithelial-mesenchymal transformation in hepatocellular carcinoma cells. To provide a new experimental basis for invasion and metastasis of liver cancer, the above hypothesis was confirmed by highthrough put gene screening, ChIP, CoIP, luciferase and so on, from the four dimensions of tissue, cell, molecule and animal.

LncRNAs在肝癌侵袭转移过程中发挥重要作用，但具体机制尚未完全明确。申请人发现将肝癌细胞敲减抑癌基因Hint1后一种新型LncRNA ETAPOD1（EMT and invadopodia-related LncRNA 1）及Dlg5表达下调，Dlg5能抑制肝癌细胞侵袭性伪足形成及上皮间质转化，且敲低ETAPOD1后促使Dlg5下调、细胞侵袭性伪足形成及上皮间质转化；生物信息筛选及荧光素酶实验证实miR-101b-3p靶向调控Dlg5。项目拟研究ETAPOD1是否通过ceRNA机制竞争性结合miR-101b-3p，从而上调Dlg5来抑制肝癌细胞侵袭性伪足形成及上皮间质转化，进而抑制肝癌侵袭转移。项目拟从组织、细胞、分子、动物四个维度，通过高通量筛查、ChIP、CoIP、荧光素酶等技术证实上述假设，为肝癌侵袭转移提供新的实验依据。

LncRNAs在肝癌侵袭转移过程中发挥重要作用，但具体机制尚未完全明确。申请人发现将肝癌细胞敲减抑癌基因Hint1后一种新型LncRNA ETAPOD1及Dlg5表达下调，Dlg5能抑制肝癌细胞侵袭性伪足形成及上皮间质转化，且敲低ETAPOD1后促使Dlg5下调、细胞侵袭性伪足形成及上皮间质转化；生物信息筛选及荧光素酶实验证实miR-101b-3p靶向调控Dlg5。项目拟研究ETAPOD1是否通过ceRNA机制竞争性结合miR-101b-3p，从而上调Dlg5来抑制肝癌细胞侵袭性伪足形成及上皮间质转化，进而抑制肝癌侵袭转移。有以下主要发现/关键数据：（1）利用miRwalk、targetscan、miRbase数据库探索建立lncRNA-miRNA-Dlg5 mRNA调控关系，发现lncRNA ETAPOD1可能通过介导miRNA-101b-3p逃避参与Dlg5调控网络。（2）检测临床肝癌标本和癌旁组织中 LncRNA ETAPOD1 的表达情况，发现癌旁组织中的LncRNA ETAPOD1 表达下降。（3）荧光素酶报告基因实验证明miRNA-101b-3p与Dlg5直接结合，并抑制Dlg5mRNA表达。（4）敲低LncRNA ETAPOD1，Dlg5 mRNA随之表达下调。（5）在HepG2细胞，过表达LncRNA ETAPOD1，侵袭性伪足形成减少。（6）发现肝癌细胞中Hint1敲低后，另一lncRNA 同样EPB41L4A-AS1异常表达，肝癌细胞系内lncRNA EPB41L4A-AS1表达明显高于永生化肝细胞系，且高侵袭性肝癌细胞系EPB41L4A-AS1表达明显高于低侵袭性肝癌细胞系。（7）临床预后分析发现：对80例肝癌患者行Kaplan-Meier曲线分析，显示EPB41L4A-AS1高表达组病例OS及DFS好于低表达组，TCGA在线数据库验证显示EPB41L4A-AS1高表达患者组DFS明显好于低表达组。（8）LncRNA EPB41L4A-AS1在体外促进HCC细胞EMT。