TAGLN介导的结直肠癌转移及HMGA2对其调控作用研究

The mechanisms that the abnormal expression of Transgelin (TAGLN) ,a member of actin-binding proteins, correlates with tumor metastasis were uncertain. Our previous study found that overexpressed TAGLN in colorectal cancer cell (CRC) induced epithelial-mesenchymal transition (EMT) and promoted colorectal metastasis. Among dozens of genes affected by TAGLN, transcription factor HMGA2 was noted and thought to induce the nuclear translocation of TAGLN. In present research, the cell lines, animal models of metastasis and clinical specimens of CRC would be employed and variety of experiments (western blot, cell migration and invasiveness assay, immunoprecipitation, immunofluorescence, et al) would be used, combined with pathways agonists and inhibitors, in order to illucidate the key pathways inducted the EMT and metastasis of CRC by TAGLN. Furthermore, the interaction between TAGLN and HMGA2 would be defined and the functions of TAGLN-binding- HMGA2 on the migration and invasiveness of CRC be extensively analyzed. This study will reveal for the first time the concrete mechanisms that TAGLN promotes CRC metastasis in vitro and in vivo and improve the comprehension of the molecular mechanisms that actin-binding proteins involve in the progress of metastasis, which would also provide novel targets for evaluation and therapies of CRC metastasis.

肌动蛋白家族成员Transgelin（TAGLN）的异常表达常与肿瘤转移相关而机制尚不明确。我们前期研究发现，结直肠癌（CRC）中TAGLN过表达、与上皮间质转化（EMT）相关、可促进肿瘤转移；且可能与转录因子HMGA2结合而导致细胞核转位。本项目拟针对CRC细胞、小鼠肿瘤转移模型及临床标本等，通过免疫印迹、侵袭和迁移实验、免疫沉淀、免疫荧光及双染色、凝胶移动迁徙率分析等方法，配合信号通路抑制剂和激动剂的使用，观测调控TAGLN的上游关键通路，明确TAGLN是否介导Akt、JNK和Wnt通路改变引起的结直肠癌EMT与转移；寻找TAGLN与HMGA2结合的确凿依据；并深入分析HMGA2对TAGLN介导的CRC侵袭和转移所发挥的调控作用。本研究将首次对TAGLN促进CRC转移的分子机制进行深入研究，拓展了对细胞骨架结合蛋白参与肿瘤转移分子机制的认识，也为CRC转移评估和治疗提供新的靶点。

背景：在世界范围内的癌症死因排序中，结直肠癌（colorectal cancer，CRC）居第二位，其中90%的死亡可归因于肿瘤转移，因此针对结直肠癌转移开展的分子机制研究对于治疗和预后至关重要。研究表明TAGLN与肝癌、胃癌、胰腺癌和结直肠癌等胃肠道肿瘤的转移关系密切，本课题探讨TAGLN介导和调控结直肠癌转移的作用及机制研究。主要研究内容：利用Wnt3a、Ly294002 或Sp600125分别处理HCT116Ctl、RKOCtl、HCT116TAGLN-KD 和RKOTAGLN 细胞，观察细胞内源TAGLN 的表达情况及细胞定位，EMT 标志蛋白的变化情况及细胞形态的变化；同时利用免疫沉淀、免疫荧光共聚焦等检测CRC 细胞中TAGLN 与HMGA2 的结合以及TAGLN上调或下调对TAGLN-HMGA2 结合的影响，EMSA检测TAGLN表达的变化对HMGA2的DNA结合能力的影响，免疫荧光双染、激光共聚焦显微镜观察小鼠肿瘤转移模型转移灶中TAGLN-HMGA2 结合情况和临床直肠癌原发灶中TAGLN-HMGA2 结合与肿瘤分期的相关性；最后通过对HMGA2表达的调控体内体外观察结直肠癌细胞转移能力、细胞克隆形成能力的变化。重要结果：TGF-β可以诱导TAGLN的表达促进结直肠细胞侵袭迁移，并且促进TAGLN核转位；而且对结直肠细胞中TAGLN下调后，TGF-β促进侵袭迁移和促TAGLN核转位的作用被逆转，同时上皮间质化行为也被抑制；而在结直肠癌细胞中过表达HMGA2则可以恢复抑制TAGLN产生的效应。 科学意义：研究表明TGF-β-TAGLN-HMGA2通路可能在结直肠癌转移的过程发挥重要作用，有望成为临床防治结直肠癌转移的新靶点。