电压门控钠离子通道Nav1.5在肝癌发生发展与靶向治疗中的作用及其机制研究

Various classes of ion channels play major roles in cancer progression. Voltage-gated sodium channel Nav1.5 has been implicated in cell proliferation, invasion and metastasis potential of many tumors. However, the functional activity of Nav1.5 and its specific contribution to tumor progression in hepatocellular carcinoma has not been delineated. We have shown previously that lidocaine induces apoptosis and suppresses tumor growth in human hepatocellular carcinoma cells in vitro and in a xenograft model in vivo. However, the mechanisms are currently unknown. Here, we demonstrated that Nav1.5 is up-regulated in HCC compared with matched normal liver tissue and the level of Nav1.5 expression correlates with their poor prognosis. Moreover, we found that lidocaine as a nonspecific voltage-gated Na+ channel inhibitor can depress the Nav1.5 expression in human HepG2 cells. These suggest that Nav1.5 may be involved in HCC progression. In this study, we will identify the expression of Nav1.5 in HCC by immunohistochemical staining and analyse the correlation to prognosis .The patch clamp technique, stable up/down-regulation of Nav1.5 and xenografts in nude mice will be used to investigate the molecular mechanisms involved in Nav1.5-induced proliferation, invasion and metastasis. In addition, we will use the specific voltage-gated Na+ channel inhibitor tetrodotoxin (TTX) to discuss the possibility of voltage-gated Na+ channel inhibitors being targeted therapy drugs in HCC. The results of this research maybe can provide some useful evidence for the pathogenesis and targeted therapy in HCC.

恶性肿瘤的发生发展有多种离子通道参与，其中电压门控钠离子通道Nav1.5的Na+电流增强及异常表达与多种肿瘤的增殖和转移密切相关，但在肝癌中尚未见报导。我们的前期研究结果表明：非特异性Na+通道阻滞剂利多卡因可抑制肝癌细胞生长，并可增强顺铂抗肿瘤效果，其机制不明。预实验结果提示：Nav1.5在肝癌组织中表达上调，且其高表达与不良预后有关；利多卡因可抑制肝癌细胞Nav1.5的表达。提示Nav1.5可能参与了肝癌的发生发展。本项目拟采用免疫组织化学法检测Nav1.5在肝癌组织中的表达，分析其作为肝癌预后标志物的意义。通过使用膜片钳、构建病毒感染的过表达/敲低稳定细胞株和裸鼠肝癌模型等实验，阐明Nav1.5的电活动和表达水平对肝癌增殖、侵袭与转移的影响及分子机制。并通过使用Na+通道阻滞剂TTX的药理学手段，探讨其成为肝癌治疗新靶点的可能性。本研究有望为肝癌的发病机制和靶向治疗提供新的理论依据。

恶性肿瘤的发生发展有多种离子通道参与，其中电压门控钠离子通道Nav1.5的Na+电流增强及异常表达与多种肿瘤的增殖和转移密切相关，但在肝癌尚未见报导。前期结果发现：Nav1.5抑制剂利多卡因可抑制肝癌细胞生长，其作用可能是抑制了Nav1.5的表达。且Nav1.5抑制剂利多卡因已被证实发挥去甲基化作用调节肿瘤细胞的增殖，且增敏顺铂。我们前期的研究结果亦表明，Nav1.5抑制剂利多卡因可提高肝细胞癌对顺铂的敏感性。本课题试图进一步厘清利多卡因作为Nav1.5抑制剂，在其发挥的抗肿瘤作用中抑制Nav1.5与DNA去甲基化是否存在内在联系。在本研究中，我们明确了肝癌组织中Nav1.5 mRNA水平明显高于癌旁肝组织或正常无肝硬化肝组织。证实了过表达Nav1.5的SMMC-7721细胞增殖率明显高于对照组SMMC-7721-Vector的增殖率；而敲低Nav1.5的MHCC-97H细胞增殖率明显低于对照组MHCC-97H-NC-shRNA的增殖率。发现了Na+通道阻滞剂（利多卡因、罗哌卡因和布比卡因）在人肝癌细胞中具有抑制肝癌细胞生长和去甲基化作用；并且这些Na+通道阻滞剂（酰胺类局麻药）可能通过上调RASSF1A表达和去甲基化在人肝癌细胞中增敏顺铂。我们的研究为进一步阐明肝癌的发病机制和靶向治疗提供了新的理论依据。