Copine-1蛋白抑制肺腺癌细胞上皮-间质转换(EMT)机制研究

Lung cancer is a malignant cancer with the highest mortality-rate worldwide. Intensive investigation is therefore urgently needed to understand the mechanisms of lung cancer and to search for specific diagnostic markers and therapeutic targets. Based on the comparative analysis of subcellular proteomics between human lung adenocarcinoma cell line A549 and human bronchial epithelial cell line HBE, copine-1 was found to be dramatically down-regulated in both A549 and SPCA-1 cancer cells. Immunohistochemistry (IHC) results suggested that the expression of copine-1 in lung adenocarcinoma tissues was associated with the TNM stage (p<0.05). To investigate the biological roles of copine-1 in lung tumorigenesis in vitro, copine-1 was respectively over-expressed in lung adenocarcinoma cell lines by retrovirus-mediated transfection and knocked-down by lentivirus-mediated shRNA technology. Stable cell lines A549/CPNE1 with copine-1 over-expression and A549/KD with copine-1 knock-down were established. Proliferation and migration assays were performed respectively and the results suggested that copine-1 functioned as a tumor suppressor in lung adenocarcinoma. Furthermore, we found the down-regulation of vimentin and up-regulation of E-cadherin in A549/CPNE1 cells, and the reverse phenomena of the protein expressions in A549/KD cells. Moreover, we confirmed that the expression level of copine-1 has an inverse correlation with cell migration induced by TGF-β1. All these observations suggested that copine-1 may play an important role in EMT and MET transformation, affecting cell proliferation and migration. The detailed mechanism underlying copine-1 mediated cell transformation, as well as the mechanism of transcriptional regulation of copine-1 need further study. It will provide potential clinical applications of molecular targeted therapy in lung adenocarcinoma.

肺癌是目前致死率最高的恶性肿瘤，进展期的肺癌目前尚无有效治疗方法。发现有效的新治疗靶标，是提高肺癌患者治愈率的关键。Copine-1是Ca2+依赖的磷脂结合蛋白，确切生物功能未知。本项目前期研究首次发现：与正常细胞相比，Copine-1在肺腺癌细胞中表达下调；且Copine-1在肺腺癌组织中的表达随患者临床分期进展而逐步下降。通过稳定过表达和敲低Copine-1都表明该分子能够抑制肺腺癌细胞的增殖和迁移，并初步阐明Copine-1参与调控细胞EMT。提示Copine-1可能是一个新的抑癌因子。本项目是在前期研究新发现的基础上，利用蛋白质组学技术、酵母双杂交、报告基因系统等实验方法，系统研究Copine-1在肺腺癌细胞中的转录调控过程、Copine-1下游调控蛋白及其与EMT的关系，旨在阐明Copine-1在肺腺癌中的抑癌机制，为深入了解肺腺癌发生发展，研发新治疗靶标提供新思路。

肺癌是严重威胁人类健康的恶性肿瘤，非小细胞肺癌的发病率占肺癌发病率的85%以上，而肺腺癌是非小细胞肺癌的主要类型之一。因此，深入研究、发现特异的肺腺癌诊断标记物和治疗靶标显得尤为重要。上皮-间质转换（EMT）是肿瘤细胞发生转移的重要初始步骤，在肿瘤浸润和远端转移中扮演了重要的角色。Copines 蛋白家族是一类磷脂结合的蛋白，具体的生物功能尚未阐明。我们在前期的差异蛋白质组学研究中发现，copines家族九个成员之——copine-1蛋白在肺腺癌细胞中的表达显著下调。可能是潜在的肿瘤抑制因子，但是其抑制肺腺癌的机制还不明确。在此基础上，我们证实copine-1是肺腺癌细胞的抑制因子，通过细胞实验和裸鼠模型，证实copine-1表达下调促进肺腺癌细胞的增殖和转移。基于细胞培养稳定同位素标记技术（SIALC）的定量蛋白质组学实验以及信号通路研究表明：Copine-1下调通过经典的TGF-β1/Smad3信号通路促进了上皮-间质转换（EMT）；copine-1表达下调，促进了TGF-β1的基因转录、蛋白质表达以及细胞外分泌，从而通过TGF-β1自分泌环激活TGF-β1信号通路；另一方面，证实了过表达copine-1逆转了上述提及的促进EMT的变化，即过表达copine-1促进细胞发生了间质-上皮转换（MET）。研究还揭示SQSTM1/P62的表达受copine-1的正调控，并且参与TGF-β1对EMT的信号通路调节。免疫组化的结果表明Copine-1 蛋白与肺腺癌患者的病理分级和临床分期相关。本研究初步阐明Copine-1 在肺腺癌中的抑癌机制，为深入了解肺腺癌发生发展，研发新治疗靶标提供新思路。