4.1N基因调控EMT在结肠癌转移中的作用及分子机制研究

Epithelial-mesenchymal transition (EMT) is a crucial initiating step in tumor invasion and metastasis. The progression of EMT involves a dramatic reorganization of the cytoskeleton. Protein 4.1 is a key membrane-cytoskeleton cross-linker and adaptor that not only can maintain cell shape and cell motility, but also plays an important role in tumor formation, progression and metastasis. In our preliminary study, it has been demonstrated the expression of 4.1N was lost in the poorly differentiated and high metastasis colon cancer cell lines and tissues, suggesting 4.1N is tightly associating with the colon cancer metastasis. Moreover, 4.1N was found to be co-localized with EMT marker, E-cadherin, in high differentiated colon cancer cell. The results of RNA sequencing of 4.1N rescued colon cancer cells revealed significantly down-regulated expression of ZEB1 and vimentin, which are key regulator and mesenchymal marker of EMT respectively, and up-regulated expression of ZEB1-associated miRNA200b. To further explore the molecular mechanisms of the involvement of 4.1N in EMT and the effects of 4.1N on colon cancer metastasis, the present project was designed to determine the influence of 4.1N in modulating the mesenchymal transition of colon cancer cell line, to examine the modulatory effects and molecular mechanism of 4.1N by interacting with ZEB and miRNAs during EMT progress, and finally to clarify the pathological significance of 4.1N in metastasis of colon cancer as a novel marker of EMT.

上皮间质转化(EMT)过程是肿瘤侵袭与转移的关键启动步骤，与膜骨架的重塑密切相关。蛋白4.1是细胞膜蛋白与骨架蛋白连接的关键分子，不仅参与维持细胞形态和运动调控，在肿瘤的发生及转移中也有重要作用。我们前期研究发现其家族成员4.1N与结肠癌转移密切相关，在结肠癌组织标本及低分化高转移的癌细胞株中4.1N表达普遍缺失。此外，在上皮型高分化结肠癌细胞中4.1N与EMT标志分子E-cadherin共定位；RNA-Seq结果显示4.1N导入缺失表达的结肠癌细胞后EMT间质标志分子vimentin与转录因子ZEB1表达均显著下降，而与ZEB1相关miRNA200b的表达升高。基于此本项目拟通过研究4.1N在结肠癌细胞间质化中的作用，探讨4.1N与ZEB-miRNAs在EMT中的相互调控关系，阐明4.1N通过EMT调控结肠癌转移的分子机制，明确4.1N作为潜在的EMT调控分子在结肠癌转移中的病理意义。

尽管既往研究发现蛋白4.1家族成员能够参调控肿瘤上皮间质转化（EMT），进而影响肿瘤转移的发生，但对其机制尚且了解极少。本项目按计划综合利用结肠癌细胞系、裸鼠皮下移植瘤和裸鼠肿瘤肺部转移等体内、外研究模型，从分子、细胞和整体动物水平系统阐明了蛋白4.1家族成员4.1N通过调控EMT进而抑制结肠癌转移的作用及分子机制。本项目研究首先通过在缺失表达4.1N的结肠癌RKO细胞中构建稳定表达4.1N的体系，证明了蛋白4.1N表达上调能够显著抑制RKO细胞的增殖、迁移和侵袭；并且能够诱导RKO细胞中上皮型细胞标志分子E-钙黏蛋白（E-cadherin）的下调，间质型细胞标志分子波形蛋白（Vimentin）及基质金属蛋白酶MMP-2和MMP-9等EMT效应蛋白的上调。基于此，深入研究其机制发现，蛋白4.1N能够通过使细胞内Rho-GTP酶RhoA激活受阻，引起其下游Raf-MEK-ERK通路中多个信号分子磷酸化水平下降，进而导致与EMT发生密切相关的重要转录因子ZEB1的表达下调以及非编码microRNA-200家族成员的表达上调。此外，本项目利用BALB/c裸鼠建立皮下移植瘤及肺转移模型，发现稳定表达蛋白4.1N的结肠癌细胞RKO在移植裸鼠后的生长和肺转移能力明显低于野生型。综上所述，本项目的研究结果阐明蛋白4.1N能够通过调控Raf-ERK-miRNA200s轴抑制结肠癌细胞的EMT进程，进而影响肿瘤细胞的生长与转移。