介入栓塞术后超级增强子源lncRNA-MET经循环exosome介导肝癌转移的作用及表观调控机制研究

Hypoxic microenvironment post transcatheter arterial chemoembolization(TACE) promotes liver cancer metastasis with unknown mechanisms. Recently, exosome are reported as key mediators in the formation of premetastatic niche. Here, we identified that exosomes isolated from sera in the liver cancer patients post TACE could significantly enhance migration of human lung microvascular endothelial cell (HMVEC) via exosomal RNA. Moreover, by RNA-seq approach, we demonstrated that most RNA molecules in the exosome are long non-coding RNAs (lncRNAs). In order to interrogate the transcriptional origin of these lncRNAs, we performed a series of ChIP-seq/RNA-seq experiments in hypoxic vs normoxic liver cancer cell line (HepG2) and integrated these dataset with the previous exosome RNA-seq data. Surprisingly, we found that the lncRNA with highest expression in exosomal RNA (namely lncRNA-MET) is derived from a super enhancer (SE) region in HepG2. Furthermore, functional studies revealed that overexpression of lncRNA-MET could downregulate ZO-1 expression, which is a gap junction protein implicated in cancer metastasis. Interestingly, JQ1, a BRD4 (SE associated protein) inhibitor in clinical trial, could dramatically downregulate lncRNA-MET. Taken together, these preliminary data shed light on the importance of investigation of functional relevance and regulatory mechanisms of lncRNA-MET in liver cancer metastasis. Therefore, in this project, we aim 1) to elucidate the effects of lncRNA-MET on liver cancer metastasis in hypoxic microenvironment; 2) to demystify the epigenetic mechanisms of exosomal lncRNA-MET in mediating liver cancer metastasis; 3) to unravel the roles, epigenetic mechanisms and clinical significances of BRD4 in the transcriptional regulation of lncRNA-MET; 4) to perform the translational studies of JQ1 for control of liver cancer metastasis post TACE. Collectively, the proposed studies will not only clarify the molecular mechanisms of liver cancer metastasis post TACE but also implicate in the molecular surveillance of liver cancer metastasis and revolutionize the current therapeutic panel by the combination of TACE+JQ1.

肝动脉化疗栓塞（TACE）术后形成的缺氧微环境能促进肝癌转移但机制不详。外泌体（exosome）是近年报道形成癌转移微环境的重要媒介。我们发现TACE术后患者循环exosome能显著增加人肺微血管内皮细胞（HMVEC）的迁移能力并主要由其中的长非编码RNA（lncRNA）介导。随后，表观基因组实验发现其中表达最高的lncRNA（lncRNA-MET）来源于缺氧肝癌细胞的超级增强子（SE）区域，并证实它可下调HMVEC中缝隙连接蛋白ZO-1的转录及受到SE相关调控蛋白BRD4的抑制剂JQ1所抑制。本课题拟进一步阐明缺氧下exosomal lncRNA-MET对肝癌转移的作用及表观遗传机制；明确BRD4调控lncRNA-MET转录的作用、表观遗传机制及临床意义；探讨JQ1控制TACE术后肝癌转移的转化医学价值。本课题将有望揭示TACE术后转移的新机制和建立“TACE+JQ1”的全新治疗模式。

原发性肝癌（primary liver cancer，PLC）是我国最常见的恶性肿瘤之一，目前以经肝动脉化疗栓塞（transarterial chemoembolization，TACE）为基础的综合治疗已成为中晚期肝癌的重要治疗模式，但 TACE 术后“易复发、高转移”的临床难题一直是导致患者远期预后不理想的重要因素，因此，探索肝癌发生发展的内部机制对于肝癌的治疗尤其重要。本项目在肝癌复发转移的表观遗传机制方面，立足于表观基因组和三维基因组核心领域“超级增强子”，我们通过高通量的测序数据获得了肝癌细胞内部深层次的染色质互作模式，并挖掘和筛查出了来源于超级增强子相关的lncRNA，通过RNA-seq\ChIA-PET深入探索了lncRNA-MET形成的机制，利用CTCF的ChIA-PET，我们发现潜在的HCC特异性CTCF结合位点和循环结构；发现特异LncRNA及miRNA均有促进肝癌转移的能力，通过研究发现miR-125b-5p及lncHOXA11-AS在肝癌及癌旁组织中表达具有差异，且miR-125b-5p通过靶向结合下游的靶基因TXNRD1抑制肝癌的增殖与转移；此外，围绕介入栓塞和缺氧微环境，我们还开展了肝癌介入研究相关exosome、CtDNA等相关的转化研究项目，建立了外泌体exosome、ctDNA等液态活检的成熟技术平台，探索了肝癌转移的分子机制，通过测序对肝癌患者外周血的ctDNA突变特征进行分析后发现，ctDNA中最常见的突变基因是TP53和TERT，ctDNA丰度与PVTT、BCLC分期有关，可作为介入治疗的预后指标，将这些患者的组织TMB和血液TMB进行相关性分析证实ctDNA可能是一种可靠的生物标志物。研究发现BRD4 抑制剂 JQ-1 显著影响超级增强子相关 LncRNA表达和肝癌的生长发展，表现出良好的临床转化潜能。以上的研究结果为临床医生选择肝切或TACE的手术方式进行决策建议，总之，本项目的研究成果覆盖在肝癌的发生发展机制、诊断、治疗手段、预后等全流程，为肝癌临床治疗的转化应用提供坚实的科学依据和理论基础。