利用CRISPR技术探索Nrf2抗氧化通路在低氧诱导肺癌转移中的作用及机制

Relative hypoxia may induce tumor metastasis by increasing intracellular oxidative stress and subsequent epithelial mesenchymal transition (EMT), thus facilitating tumor invasion and migration. Nrf2 transcription factor mediated antioxidant signaling pathway is the most important endogenous protective mechanism against oxidative stress and Nrf2 gene silencing may induce tumor metastasis as a result of elevated intracellular oxidative stress. Our previous results show that Nrf2 mediated antioxidant signaling is inhibited by hypoxia, which may work as another underlying mechanism of the hypoxia induced tumor metastasis. Moreover, NRF2 activating mutation is associated with improved survival in early stage non-small cell lung cancer patients after curative surgery. In the present study we aim to further verify the role of Nrf2 associated antioxidant signaling in tumor metastasis induced by hypoxia and test the feasibility of upregulating endogenous antioxidant by introducing NRF2 activating mutation through CRIPSR/CAS9 gene editing tool to suppress lung cancer metastasis, which may contribute to the development of targeted gene therapy against this pathway in future.

低氧通过升高细胞内氧化应激水平诱导肺癌细胞上皮间质转化，使其侵袭迁移能力增加从而促进肿瘤转移。转录因子Nrf2是细胞内最重要的抗氧化机制，但迄今对Nrf2在低氧诱导的肿瘤转移中的作用仍知之甚少。我们前期研究结果发现低氧可以抑制Nrf2相关抗氧化物表达使氧化应激水平增加，而含NRF2基因激活突变的早期非小细胞肺癌患者手术后预后较好。因此我们提出假说：Nrf2抑制可能是低氧促进肿瘤转移的机制之一，而NRF2激活突变可以减轻低氧诱导的肿瘤转移。本项目旨在进一步验证这一假说，并利用CRISPR/CAS9基因修改工具在肺癌细胞内引入NRF2基因激活突变，以探索通过基因修改上调内源性抗氧化物水平抑制肿瘤侵袭及转移的可行性，为未来针对这一机制的基因治疗寻找初步依据。

肺癌是目前发病率及致死率最高的恶性肿瘤，其预后取决于转移程度。低氧是诱导肺癌转移最重要的因素之一。在本研究中，我们在手术切除标本中发现肿瘤缺氧程度与肺癌恶性程度及淋巴结转移状态存在相关性，且实验室低氧可以诱导肺癌细胞表皮间质转化，提高其增殖迁移能力。同时，肺癌的病理亚型与淋巴结转移风险及缺氧程度可能存在相关性，术中病理亚型判断可能用来指导肺癌选择性淋巴结清扫及微创手术方式。另一方面，在肺腺癌细胞中，低氧能够抑制Nrf2信号通路，且NRF2基因突变与肺鳞癌患者术后生存存在一定相关性。然而，刺激Nrf2信号通路并不能逆转低氧引起的肺癌转移。在本研究中，我们也探索了低氧对于肺癌细胞中环状RNA的影响及脂肪代谢通路的影响。低氧能显著降低肺癌细胞中hsa_circ_0008193的表达，且在临床标本中hsa_circ_0008193的降低水平与肺癌缺氧程度及淋巴结转移状态相关，提示其可能作为判断肺癌缺氧程度及转移风险的生物学标志物。此外，在脂代谢相关转录因子C/EBP在肺腺癌中显著升高，这两项发现的临床价值及潜在机制均值得进一步探索。