EGFL7转录激活在肝癌介入栓塞后侵袭转移中的作用及机制研究

Trans-catheter arterial chemo-embolization (TACE) is the main strategy for treatment of hepatocellular carcinoma (HCC) with BCLC stage B. However, enhancement of metastasis after TACE is the bottleneck to limit its effect. This is associated with several gene transcriptional activation induced by hypoxia due to TACE (mainly due to trans-catheter arterial embolization, TAE). Epidermal growth factor-like domain 7 (EGFL7) has been discovered recently as an angiogenesis factor, taking similar structure of Notch receptor area, but has been rarely reported in hepatocellular carcinoma. Our previous study found that patients with HCC treated with TACE showed increased transcription activation and expression of EGFL7 in peripheral blood, which is associated with tumor metastasis. The invasive and angiogenic capabilities are significantly weakened by EGFL7 antibody in HCC cells. Accordingly, we initiated the hypothesis: EGFL7 transcriptional activation occurs in HCC after TACE, and promotes the metastasis of HCC. Notch pathway is one of the possible mechanisms involved in this process. This project aims to investigate the influence of TAE on the expression of EGFL7 by observing the changes of EGFL7 protein from blood samples of HCC patients treated with TACE, and changes of EGFL7 mRNA and protein in pathological sections after TAE in animal models. With human HCC cell and animal models, using gene silencing and over-expression to target the regulation of EGFL7 expression in order to block the Notch pathway, this project also aims to explore the relationship between EGFL7 and metastasis of HCC generally on molecules, cellular, histic and animal levels. This project will provide theoretical and experimental basis for EGFL7 as a potential target for HCC gene therapy, which could be combined with TACE as a new comprehensive treatment for HCC patients.

肝癌介入栓塞后侵袭转移的增强是限制其疗效的瓶颈，迄今对这一现象的机制仍知之甚少。EGFL7是新发现的受缺氧调控的促转移基因，结构类似Notch通路受体功能区。我们的前期研究提示，肝癌患者介入栓塞后血清EGFL7显著升高，兔肝癌模型肝动脉栓塞（TAE）后EGFL7表达上调，EGFL7抗体阻断后肝癌细胞侵袭能力减弱。据此假说：肝癌TAE后EGFL7表达上调，促进其侵袭转移，Notch通路是重要的参与机制。为验证该假说，我们应用ELISA、实时定量PCR、Western blot从临床与基础、体内与体外研究TAE对肝癌EGFL7表达的影响。用慢病毒转染、RNA干扰调节人肝癌细胞EGFL7表达，适时阻断Notch通路，从分子、细胞、组织及整体水平研究EGFL7在肝癌侵袭转移中的作用，并探讨可能机制。本研究将从EGFL7这个新视点揭示肝癌介入栓塞后侵袭转移增强的现象与机制，为肝癌靶向治疗提供新思路。

肝动脉化疗栓塞（TACE）是治疗肝癌的重要方法，在我国肝癌治疗中具有举足轻重的地位。然而，TACE具有不彻底性，且TACE导致的缺氧可促进残癌组织恶性进展。目前，关于TACE对肝癌生物学特性影响的研究很少，不足以解释肝癌介入栓塞后恶性进展的机制。EGFL7是新进发现的促癌基因，在多种实体瘤中高表达，并与侵袭转移和不良预后相关。.本课题组按照原定计划开展研究，并在此基础上优化了研究方案。（1）在表象研究中，课题组通过临床标本、缺氧培养的肝癌细胞，证实了介入栓塞导致的缺氧可促进EGFL7表达上调。（2）在效应研究中，课题组采用基因沉默和过表达技术，调节肝癌细胞株EGFL7的表达，在离体细胞和裸鼠肝脏原位模型中，确证了EGFL7具有促进肝癌恶性进展的作用。.在机制研究中，课题组注意到一个关键蛋白：细胞周期蛋白依赖性激酶调节亚基（Cyclin‐dependent kinases regulatory subunit 2 ,CKS2）。CKS2 是β-catenin的靶基因，与结直肠癌细胞高转移迁移负担有关。故本课题组接下来主要研究了EGFL7 通过Wnt/β-catenin 信号通路，进一步调控下游CKS2 的表达，从而参与肝癌的发生发展。我们采用过表达和 RNA i 沉默进行研究，检测Wnt/β-catenin 信号通路相关蛋白的表达情况，发现过表达EGFL7后，β-catenin, CKS2, CDK2 的表达明显上调，干扰EGFL7后，β-catenin, CKS2, CDK2 的表达明显下调。干扰CKS2表达后显著抑制EGFL7过表达诱导的细胞增殖，CKS2、CDK2及caspase-3的表达均下调。这些结果提示：EGFL7 在肝癌发生发展中的作用，可能与 Wnt/β-catenin 信号通路调控有关。.综上，本课题以介入栓塞为切入点，EGFL7 为主线，人肝癌细胞及裸鼠原位模型为研究对象，Wnt /β-catenin信号通路为机制新视点，证明了EGFL7可通过EGFL7-β-catenin轴促进肝癌的进展，进一步调控下游 CKS2 表达，尤其在肝癌细胞侵袭迁移进程中发挥重要作用。本项目从 EGFL7 这个新视点丰富了肝癌发生发展的分子机制，有望为肝癌综合治疗提供了潜在的新靶点。