肿瘤间质细胞lncRNA-SC1对血管生成拟态的调控及其在肝癌复发转移中的作用研究

Vasculogenic mimicry (VM) is a new model of tumor angiogenesis, which is an important reason for hepatocellular carcinoma (HCC) relapse and metastasis. Long non-coding RNA (lncRNA) can regulate the expression of protein-coding genes at both transcriptional and post-transcriptional level and its uncontrolled expression may decide tumor development and proprognosis. However, there is no direct research on the question that whether lncRNA in the tumor mesenchymal cells has regulating effect on VM and the relapse and metastasis of tumor. In our previous research, we found lncRNA-SC1 are closely related to the formation of VM in HCC. When lncRNA-SC1 was knocked down, not only the formation of VM was reduced but also the migration of HCC cells was inhibited. These evidences support to predict that lncRNA in the tumor mesenchymal cells may have regulating effect on VM and could promote the relapse and metastasis of HCC through changing the biological behavior of tumor cells. In this project, we will center on this hypothesis and aim to identify the truth that lncRNA-SC1 is highly expressed specifically in HCC mesenchymal cells and to explore the function and mechanism of lncRNA. Furthermore, we will assess the potential role of lncRNA-SC1 as a target for both prognosis evaluation and treatment against HCC recurrence and metastasis and therefore provide a new target for prognosis monitoring and targeted therapy of HCC.

血管生成拟态(VM)是一种全新的肿瘤内血管生成模式，VM的存在是肝癌术后复发转移的重要原因。长链非编码RNA(lncRNA)能够在转录及转录后水平调节蛋白编码基因的表达，其表达失控可决定肿瘤的进展及预后。肿瘤间质细胞中的lncRNA的作用，尤其是对VM及肝癌的复发转移是否有调控作用，目前还尚无直接研究。在前期工作中，我们发现lncRNA-SC1与肝癌VM形成关系密切，敲低lncRNA-SC1后，不仅能够减少VM的形成，而且能抑制肝癌细胞的迁移。这些证据支持我们推测：肿瘤间质细胞lncRNA-SC1可能对VM具有调控作用，并可能通过影响肿瘤细胞的生物学行为而促进肝癌的复发转移。本项目将围绕这一假说，开展对lncRNA-SC1在肝癌间质细胞中特异性高表达的确证、功能及机制研究，并评估其是否可作为肝癌复发转移的预测指标和干预靶点，从而为肝癌的预后监测和靶向治疗提供新的靶标。

在前期工作中，我们分选了HCC 患者癌和癌旁组织的间质细胞，提取了总RNA，采用Arraystar 公司的芯片，分别进行了lncRNA 和mRNA 表达谱差异分析，发现两群细胞具有显著的lncRNA 与mRNA 表达差异。对比癌旁组织，在HCC 患者癌组织间质细胞中共同表达上调的lncRNA 有583 个，我们对芯片上调的前11 位lncRNA，设计了siRNA，然后进行RNAi Screen，转染siRNA，检测血管生成拟态形成的情况，发现敲低了lncRNA-SC1 后，VM形成显著受抑制，表明lncRNA-SC1 能够正向调控肿瘤血管拟态形成。我们用RT-PCR检测的lncRNA-SC1在肿瘤组织中的表达，使用RNA原位杂交实验方法检测不同分级分期的病人组织中lncRNA-SC1的表达情况，使用基因敲除分别沉默Huh7和HepG2中的shlncRNA-SC1-1, shlncRNA-SC1-2和shlncRNA-SC1-3，发现肿瘤组织中lncRNA-SC1呈显著性高表达，验证了表达量随肿瘤组织的浸润程度（分期）与分化程度（分级）正相关，lncRNA-SC1的表达水平与肿瘤预后成负相关，与复发、增殖能力成正相关。我们用lncRNA-SC1高表达质粒及siRNA质粒转染肿瘤间质细胞，验证了lncRNA-SC1能够调控肿瘤的生长、迁移与微血管生成。综上所述，可用来评估lncRNA-SC1 是否可作为肝癌复发转移的预测指标和干预靶点，从而为肝癌复发转移的监测和靶向治疗提供新的靶标。