肝癌射频消融术后肿瘤干细胞源性exosomalLncRNA-PMN促进肺转移前微环境形成的机制研究

Radiofrequency ablation (RFA) is an important treatment for hepatocellular carcinoma (HCC). However, the "residual cancer" often causes the lung metastasis which affects the long-term effect. Recent studies have shown that the formation of lung pre-metastatic niche (PMN) mediated the process of lung metastasis, and exosome derived from the primary lesion was the “key medium” which induced the formation of lung PMN, but underlying mechanisms are unknown. We recently established a heat shock model in vitro to simulate the microenvironment after RFA, and isolated a kind of cell species named liver stem cells (LSC) with self-renewal ability and differentiation potential . After high throughput sequencing, we found that heat shock LSC secretes the exosome which was rich in LncRNA-PMN, and participates in the regulation of lung PMN formation. Hence we speculate that exosomal LncRNA-PMN (ELP) may be involved in the process of lung metastasis after RFA for HCC. Overall, in this proposal, we aim to: (1) explain the detail function and the upstream regulation mechanism of exosomal LncRNA-PMN from LSC in the process of lung PMN formation. (2) use animal model to investigate the intervention strategy of lung metastasis after RFA surgery by targeting LncRNA-PMN. This innovative project will reveal the new molecular mechanism of "residual cancer" progress into lung metastasis after RFA, and optimize the HCC curative strategies in order to improve the long-term prognosis of the HCC patients.

肝癌射频消融(RFA)术后“残癌”的肺转移影响远期疗效，研究表明转移前微环境(PMN)的形成是肿瘤发生肺转移的重要途径。原发病灶来源的外泌体(exosome)是诱导PMN形成的“关键媒介”，但具体机制不详。前期我们用“热休克”处理来模拟RFA术后病理微环境，以癌组织中一类具有自我更新和分化潜能的细胞—肝癌干细胞(LSC)为切入点，发现热休克的LSC分泌的外泌体可调控肺PMN形成的关键指标，全转录组测序后发现其内容物中显著富含LncRNA-PMN。故我们推测exosomal LncRNA-PMN(ELP)可能参与了肝癌RFA术后肺转移的过程。本项目拟：(1)研究LSC来源的ELP对肺PMN形成的作用及上游调控机制；(2) 基于兔肝癌模型探索RFA术后靶向ELP作为肺转移干预手段的价值。本创新课题有望揭示肝癌RFA术后“残癌”肺转移的全新分子机制和优化其临床干预策略，从而改善患者的远期预后。

肝癌射频消融(RFA)术后“残癌”的复发转移影响远期疗效，研究表明转移前热刺激微环境的形成可能是是肿瘤发生转移的重要诱因。本研究建立体外模拟射频消融术的细胞热刺激模型，首先观察了热刺激对肝癌细胞的影响，发现HSP40是肝癌细胞内响应热刺激最明显的蛋白质一，且抑制HSP40的表达后，在同样的热刺激条件下，肝癌细胞更容易出现凋亡，并且迁移能力下降，进一步对热休克处理的两种肝细胞系进行转录组测序发现，miR-19b-3p在热休克处理后的细胞中显著低表达，且其可以靶向结合HSP40的3’-UTR端，对HSP40发挥抑制性的作用，利用miR-19b-3p的模拟物对其进行过表达处理后发现，HSP40表达下调，肝癌细胞的凋亡水平增加，而迁移能力降低，这些研究结果创新性的解释了肝癌射频消融术后残癌复发的可能机制，为肝癌的药物与手术的联合治疗提供的可行的方法。此外，本项目对转录数据深入分析，进一步发现其他非编码RNA在肝癌进展的机制中也发挥重要作用，lncRNA PCAT6通过抑制miR-330-5p发挥促进细胞增殖和侵袭的作用。因此，本项目还表明lncRNA PCAT6可能是是肝内胆管癌发生发展的重要促癌因子之一。还发现miR-125b-5p通过靶向对TXNRD1的抑制作用，这使得miR-125b-5p可能成为肝癌临床治疗的靶点。我们还发现miR-135b是Hippo途径的一个转录靶点。基于这些数据，我们提出MST1-YAP-miR-135b的正反馈轴与HCC的恶化有关。我们的研究不仅加深了对Hippo通路内稳态的认识，而且提示miR-135b是肝癌潜在的预后生物标志物和治疗靶点。通过本项目，发现了肝癌治疗的潜在靶点，为肝癌基础研究向临床转化提供了可参考的精准转化策略。