胎肝特异性长链非编码RNA调控肝发育、肝干细胞分化和肝癌细胞上皮-间质转化的表观遗传学机制研究

Embryonic liver development is a fine-tuning process. Genes in this process are the opening and closing orderly, there are evidences indicating that some genes with high expression in fetal liver are also highly expressed in hepatocellular carcinoma and some signaling pathways exist both in fetal liver and hepatocellular carcinoma. In the early stage of this project we have found a large number of lncRNAs with high expression specificly in fetal liver, some of which have been comfirmed upregulated or downregulated in hepatocellular cacinoma, such as Pvt1 and H19. The roles of some lncRNAs in the epigenetic regulation mechanism are also unveiled. On the basis above, we intend to pick out the lncRNAs which relate to liver development, hepatoblast differentiation and hepatocarcinoma stem cell from our chip results by using the materials of cultured hepatic stem cells, embryonic stem cells, induced mesodermal cells and hepatocyte-like cells induced from embryonic stem cells in vitro. Then in order to find some more sensitive markers of liver cancer and targets against liver cancer, we plan to investigate their function in epigenetic regulation by utilizing the methods of gene clone, RNA interference, ChIP and RIP in hepatocytes, hepatic stem cells, induced hepatocyte-like cells, lncRNA gene knockout cells and lncRNA gene knockout mice.By now, no similar research was reproted through bibliographic retrieval.

胚胎期肝脏发育是一个被精细调控的过程，基因在这个过程中被有规律的开放和关闭，有证据表明许多胎肝期特异性高表达的分子在肝癌组织中上调表达，胚胎期肝脏和肝癌具有共同调控通路，基于以上分析，本课题前期用芯片技术连续观察了肝脏各发育阶段中lncRNA和mRNA表达，发现大量胎肝特异性高表达lncRNA，其中H19、Pvt1等在肝癌组织中的异常表达被确认，其参与调控的表观遗传学机制也部分被揭示，在此基础上，本课题拟利用肝干细胞、胚胎干细胞及其诱导生成的中胚层和肝样细胞，从候选lncRNA中筛选出与肝发育-肝干细胞分化-肝癌干细胞生成相关的核心lncRNA，利用基因克隆、RNA干扰、ChIP、RIP等技术在体外培养的肝细胞、肝干细胞、诱导的肝样细胞及lncRNA基因敲除细胞、小鼠中深入研究核心lncRNA参与的表观遗传学调控机制，为肝癌诊断找到更敏感检测指标，为肝癌治疗确定新靶点。以上内容经检索无报道

肝细胞肝癌（hepatocellular carcinoma, HCC)是世界范围内常见和致死率高的肿瘤之一，其中，肝癌干细胞的存在，及肝癌的肝内和肝外转移是HCC高复发率及较差预后的主要原因。最近研究发现，许多胎肝期特异性高表达的分子在肝癌组织中上调表达，胚胎期肝脏和肝癌具有许多共同调控通，这些在胎肝期和肝癌组织中共同高表达的分子在肝癌的发生发展，以及肝脏的发育中发挥着重要的作用。本课题建立了构建了DEN诱导的小鼠肝癌模型，TGF-β诱导的肝癌细胞EMT模型，收集了小鼠发育不同阶段的肝脏样本，分别通过长链非编码RNA（lncRNA）芯片的高通量筛选，获得了参与调控肝癌细胞EMT过程、涉及肝脏发育和肝癌发生发展的lncRNA，并深入地研究了lncRNA-ATB、lncRNA-PVT1、lncRNA-PXN-AS1等lncRNA在肝癌发生发展中发挥的表观遗传学调控作用。研究中发现，在TGF-β诱导肝癌细胞发生EMT过程中，lncRNA-ATB的表达显著上调，且进一步研究证实，lncRNA-ATB可通过竞争性的结合miR-200a家族，进而调控肝癌细胞内ZEB1和ZEB2的转录、表达，最终起到促进肝癌细胞发生上皮间质转化，推动肝癌的发生发展。相关研究发表在Cancer cell杂志，是首篇lncRNA对肝癌上皮间质转化中调控作用的SCI论文，相关研究成果为阐明肝癌发生发展的表观遗传学调控机制提供了重要的数据支持。此外，本课题研究过程中还发现，在胎肝中特异性高表达的lncRNA-PVT1在肝癌组织中的表达也显著上调，具有癌胚特性，PVT1可通过结合NOP，促进细胞增殖，维持肝癌细胞的干性特征，相关研究发表在肝病领域的权威杂志Hepatology上。研究确认印记基因lncRNA-H19在肝再生过程中上调表达，可抑制正常肝细胞的过度再生，维持肝脏的大小，相关研究发表在FEBS Letter杂志。