蟾毒灵干预ATP1A1-CA2轴参与调控肝癌侵袭转移作用研究

Recurrence and metastasis is one of the major factors affecting the prognosis of hepatocellular carcinoma (HCC) and it lacks effective drugs for treatment. Bufalin has been confirmed to inhibit Na+/K+-ATPase (NKA), however the mechanism of the antitumor effect of bufalin via NKA remains unclear. Our previous study demonstrated that Na+/K+-ATPase α1 (ATP1A1) was a major target of bufalin. Carbonic anhydrase 2 (CA2) was the downstream molecule of ATP1A1. It has been confirmed ATP1A1 promoted hepatocellular carcinoma (HCC) metastasis via downregulating CA2. Therefore, we propose that bufalin could inhibit HCC invasion and metastasis via modulating ATP1A1-CA2 pathway. In this project, by using shRNA methods in vitro and orthotopic liver model in vivo, we intend to investigate the correlation between the metastasis-inhibiting effect of bufalin in HCC and ATP1A1 and CA2. We will further ascertain the related pathways and their correlation with ATP1A1 and CA2 using chip technology to explore the molecular mechanisms of the antimetastatic property of bufalin. Thus, the study aims to provide theoretical basis for acertaining biomarkers reflective of bufalin’s sensitivity and promote the clinical application of cardiac glycoside against HCC.

肝癌复发转移是影响疗效的重要因素，且缺乏有效治疗药物。蟾毒灵为Na+/K+-ATP酶抑制剂，但Na+/K+-ATP酶介导蟾毒灵抑制肝癌侵袭转移的机制有待明确。我们前期研究结果表明Na+/K+-ATP酶α1亚型（ATP1A1）是蟾毒灵重要的结合位点，碳酸酐酶2（CA2）为ATP1A1下游关键分子，过表达ATP1A1蛋白可通过下调CA2蛋白促进肝癌转移。因此，我们设想蟾毒灵可通过靶向ATP1A1-CA2轴发挥抗肝癌侵袭转移作用。本课题将以ATP1A1过表达和CA2敲减的人肝癌细胞及其构建的人肝癌原位移植瘤动物模型为研究对象，从ATP1A1-CA2轴切入，解析蟾毒灵抗肝癌侵袭转移效应与ATP1A1、CA2蛋白的相关性，并通过芯片技术筛选相关信号通路，明确蟾毒灵通过ATP1A1抑制肝癌转移作用机制，为寻找预测蟾毒灵敏感性的生物标志物提供理论依据，进一步推进以蟾毒灵为组分的强心苷药物的临床应用。

本研究项目首先在体外细胞实验中确定蟾毒灵可下调肝癌细胞中ATP1A1并上调CA2表达，同时ATP1A1及CA2的表达会反向影响蟾毒灵的药物敏感性。基于此，我们通过一系列功能试验证实CA2可通过影响ATP1A1表达量进而影响蟾毒灵的药物敏感性，并通过调控EMT影响肝癌细胞的侵袭迁移能力。进一步，我们通过测序联合生物信息学分析深入挖掘并锁定蟾毒灵干预ATP1A1/CA2调控肝癌脂质代谢信号通路，并通过调控脂质合成途径中关键酶FASN/SREBP-1/ACLY的表达影响肝癌细胞的侵袭迁移与增殖能力，此外，蟾毒灵与脂代谢抑制剂的竞争性拮抗作用亦反向证明蟾毒灵与脂质代谢的密切相关性，并在体内实验中得到一致性结论。这一结果进一步阐明了蟾毒灵抑制肝癌侵袭转移的深层机制与作用环节，为蟾毒灵在肝癌复发转移防治上提供理论基础和应用的细胞分子病理生理学依据，亦为提高蟾毒灵药物敏感性及临床疗效提供了新的视角。