RCAN1.4通过抑制CaN/NFAT信号通路阻遏肝癌转移的分子机制

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent intrahepatic and extrahepatic metastasis. Active angiogenesis and metastasis are responsible for rapid recurrence and poor survival of HCC. However, mechanisms underlying metastasis and angiogenesis in HCC remain to be revealed. RCAN1 is an endogenous regulator of Calcineurin/NFAT signaling activity, and recently is reported to suppress VEGF-mediated angiogenic signaling in endothelial cells. Our preliminary results showed that expression level of isoform RCAN1.4 was significantly down-regulated in tumor tissue compared with peritumoral tissue in HCC and its expression was inversely correlated with microvessel density in HCC samples. In addition, we found that RCAN1.4 could suppress migration and invasion of HCC in vitro. Therefore, we propose that RCAN1.4 may has antitumoral effects on HCC, which may be due to its ability to regulate tumor angiogenesis of HCC. This project aims to study regulatory effects of RCAN1.4 on the metastasis and tumor angiogenesis of HCC by HCC cells, xenograft mouse models of HCC, RCAN1.4 liver-specific knockout mice, and analysis for samples from HCC patients. We also attempt to identify target molecules that are involved in antitumoral effects of RCAN1.4, clarify the roles of CaN/NFAT signaling which is regulated by RCAN1.4, and provide a potential approach for antimetastatic therapy of HCC by anti-angiogenesis.

肝癌是典型的富血管肿瘤，极易发生肝内和肝外转移。肝癌患者的预后差很大程度上是由于异常活跃的肿瘤血管生成和转移所致。然而当前对于肝癌的血管生成和转移机制仍知之甚少。RCAN1是CaN/NFAT信号通路的内源性抑制因子，据文献报道RCAN1可抑制血管内皮细胞对VEGF血管生成信号的响应。我们前期的研究结果表明，其选择剪接体RCAN1.4表达水平在肝癌组织显著低于配对的癌旁组织，且与肿瘤血管密度存在显著的负相关。在体外实验中，我们还发现RCAN1.4可以抑制肝癌细胞的迁移和侵袭。本课题计划采用HCC细胞、HCC荷瘤裸鼠和RCAN1.4肝特异性敲除鼠模型，并结合临床样本分析，探索RCAN1.4对肝癌肿瘤血管生成和转移的调控机制。通过确定介导RCAN1.4抑癌效应的关键靶分子，阐明RCAN1.4调控CaN/NFAT信号通路抑制肝癌转移的分子机理，期望为抗肿瘤血管生成治疗肝癌转移提供潜在靶点。

肝癌是典型的富血管肿瘤，极易发生肝内和肝外转移。肝癌患者的预后差很大程度上是由于异常活跃的肿瘤血管生成和转移所致。然而当前对于肝癌的血管生成和转移机制仍知之甚少。根据流行病学统计数据显示，唐氏综合征患者（又称21三体综合征）得实体瘤的概率显著低于正常人。这提示人类21号染色体上可能存在重要的抑癌基因。根据该线索我们通过Affymetrix HG-U133 Plus 2.0表达谱芯片系统分析了14对配对的肝癌组织中位于21号染色体上的差异表达基因，通过定量PCR检测了对108对肝癌组织及配对癌旁组织检测了RCAN1三种选择剪接体的表达变化，同时我们还对肝癌组织芯片（n=196）做免疫组化检测，并分析了RCAN1的表达量与肝癌患者预后的相关性。结果发现RCAN1的选择剪接体RCAN1.4的mRNA水平和蛋白水平在肝癌中均显著表达下调，而RCAN1的低表达与肝癌患者的预后不良显著相关。在体外功能实验中，发现过表达RCAN1.4可抑制肝癌细胞的增殖、运动和侵袭能力，而干扰RCAN1.4则促进肝癌细胞的增殖、运动和侵袭。在进一步的体内实验中，我们发现过表达RCAN1.4可抑制异种移植瘤的血管新生、生长和转移。进一步的分子机制研究发现，RCAN1.4通过Calcineurin/NFAT1信号通路调控IGF1和VEGFA的表达。而使用Calcineurin酶抑制剂也可以抑制肝癌细胞中NFAT1的核转录和肿瘤转移。甲基化测序结果显示RCAN1基因启动子区CpG的高甲基化导致了其在肝癌中的表达下调。我们的项目确定了介导RCAN1.4抑癌效应的关键靶分子，阐明RCAN1.4调控CaN/NFAT信号通路抑制肝癌转移的分子机理，期望为抗肿瘤血管生成治疗肝癌转移提供潜在靶点。