肌动蛋白结合蛋白TAGLN2在甲状腺乳头状癌侵袭转移中的作用及机制研究

Our previous NSFC project(81072182) demonstrated that actin-binding protein TAGLN2 is the downstream target gene of the SDF-1-CXCR7 biology axis. Further studies showed that the level of TAGLN2 increased significantly in papillary thyroid carcinoma as compared with the corresponding peritumoral normal thyroid tissues, and positively correlated with lymph node metastasis and invasion beyond the thyroid capsule. TAGLN2 konckdown in K1 cell resulted in decreasement of pseudopodia, actin microfilaments and stress fiber, inhibited cell proliferation, migration and invasion, promoted cell apoptosis. The above results suggested that TAGLN2 was related with invasion and metastasis in PTC and might participate in the process of EMT. However, the functional role and biological mechanism of TAGLN2 in PTC remained unclear. This project will investigate the influence of TAGLN2 on actin cytoskeleton remodeling and EMT, upstream regulatory mechanism of TAGLN2 expression,downstream target gene and interacting proteins of TAGLN2 by cell and animal models, and illustrated mechanisms of TAGLN2 involved in PTC invasion and metastasis. These finding may provide the scientific basis on novel biomarker of monitoring metastasis and relapse and treatment target of PTC.

我们在前项国家自然科学基金课题(81072182)研究中发现，肌动蛋白结合蛋白TAGLN2是SDF-1-CXCR7生物学轴的下游靶基因。进一步研究发现TAGLN2在甲状腺乳头状癌（PTC）组织中表达明显高于癌旁组织，且TAGLN2的表达与淋巴结转移、甲状腺被膜外侵犯相关；敲减甲状腺乳头状癌K1细胞TAGLN2的表达后，细胞表面的伪足减少，肌动蛋白微丝明显减少，应力纤维形成减少，细胞凋亡增多、细胞增殖抑制以及迁移、侵袭能力下降。以上结果提示TAGLN2与PTC侵袭转移密切相关，并且可能参与PTC的EMT过程。本项目通过细胞、动物实验及临床研究，探讨PTC中TAGLN2对肌动蛋白细胞骨架重构及EMT的影响、TAGLN2表达的上游调控机制、下游的靶基因及TAGLN2相互作用的蛋白，阐明TAGLN2参与PTC侵袭转移的分子机制，为PTC转移、复发的监测和治疗提供新的肿瘤标志物。

作为甲状腺癌中最常见的病理类型，大多数甲状腺癌乳头状癌（PTC）患者的预后较好，但仍有一部分PTC容易出现颈部淋巴结转移，严重影响患者的生存质量。本课题研究了肌动蛋白结合蛋白TAGLN2在PTC侵袭转移中的作用和分子机制。本研究发现，与癌旁组织相比，TAGLN2蛋白和mRNA在PTC组织中均表达增高，且与PTC发生颈部淋巴结转移密切相关。接下来，通过基因转染技术构建稳定表达TAGLN2-cDNA 和TAGLN2-shRNA的PTC细胞株，应用MTS、划痕、Transwell侵袭、管腔生成、细胞黏附等实验研究TAGLN2表达改变对PTC细胞功能的影响，结果提示TAGLN2能够调控PTC细胞的增殖、迁移、侵袭、血管生成及黏附能力：TAGLN2过表达促进PTC细胞增殖，提高细胞迁移、侵袭、血管形成及细胞黏附能力；TAGLN2表达下调则抑制PTC细胞增殖，降低细胞迁移、侵袭、血管形成及细胞黏附能力。然后，通过双荧光素酶报告基因实验发现miR-613能够与TAGLN2 3’UTR区结合并调控TAGLN2表达。利用免疫沉淀联合质谱筛选出TAGLN2互作蛋白，包括ACTB、MYH9、ARP-2、ARP-3等。经过基因组学和蛋白质组学结果的整合分析，当TAGLN2表达改变后，共有80个指标在基因和蛋白水平均发生了改变，其中16个指标在基因和蛋白水平均发生上调，另64个指标在基因和蛋白水平均发生下调。最后通过生物信息学分析和western blot验证，表明TAGLN2分别通过TGFβ/Smad2/c-fos通路，NRP1/PI3K/AKT/LAMC2通路和Rap1/ITGB5/FAK通路对PTC细胞的增殖、侵袭、黏附、管腔生成等功能发挥重要的调节作用。