FN1 Shapes the Behavior of Papillary Thyroid Carcinoma through Alternative Splicing of EDB Region

Papillary thyroid cancer (PTC) is often characterized by the indolent behavior with slow cell proliferation, however, small tumors still have a tendency to metastasize to the cervical lymph node, and the molecular mechanisms underlying that remain poorly understood. FN1 is an extracellular matrix (ECM) protein with the controversial role in tumors, containing three alternative splicing domains. In this study, FN1 was as the hottest gene of PTC and distinctive expression in PTC cells. The alternatively splicing EDB region of FN1 was exclusively expressed in tumors, which impacted on integrin β1 (ITGB1) bonding FN1 and it secretion process, resulting in completely distinct roles of two isoforms of FN1 including and skipping EDB domain. EDB(-)FN1 intracellularly inhibited tumor proliferation, whereas extracellular EDB(+)FN1 activated migration, invasion and lymphoangiogenesis in vitro and in vivo. Mechanistically, EDB(-)FN1 upregulated p21 in p53 signaling pathway, however, EDB(+)FN1 secretes into ECM promoted VEGFC expression. Collectively, FN1 is a potential determinant behind the characteristic behavior of PTC with its two isoforms playing distinct roles owing to the alternative splicing of EDB domain. To investigate the function of FN1 different isoforms related to EDB domain in PTC, we established K1 cell lines in which FN1 has been knocked down by shRNA, and selected knockdown monoclonal cell, then, cultured EDB(+)FN1 and EDB(-)FN1 overexpression cells by lentivirus infection.

甲状腺乳头状癌（Papillary thyroid cancer, PTC）通常以惰性生物学行为及缓慢的细胞增殖为特征，但小型肿瘤仍存在转移至颈部淋巴结的倾向，其背后的分子机制仍尚不明确。纤连蛋白1（FN1）是一类在肿瘤中作用存在争议的细胞外基质（extracellular matrix, ECM）蛋白，包含三个可变剪接结构域。本研究中，FN1被鉴定为甲状腺乳头状癌的核心差异表达基因，且在PTC细胞中呈特异性表达。FN1的可变剪接EDB结构域仅在肿瘤组织中特异性表达，该结构域可影响整合素β1（integrin β1, ITGB1）与FN1的结合以及FN1的分泌过程，进而使得携带与缺失EDB结构域的两种FN1异构体发挥完全不同的生物学功能。其中，缺失EDB结构域的FN1（EDB(-)FN1）可在细胞内抑制肿瘤增殖；而细胞外的携带EDB结构域的FN1（EDB(+)FN1）则可在体内外促进细胞迁移、侵袭及淋巴管生成。从机制层面分析，EDB(-)FN1可通过p53信号通路上调p21的表达；而分泌至细胞外基质的EDB(+)FN1则可促进VEGFC的表达。综上，FN1是决定甲状腺乳头状癌特征性生物学行为的潜在关键调控因子，其两种异构体因EDB结构域的可变剪接而发挥截然不同的功能。为探究FN1不同EDB结构域相关异构体在甲状腺乳头状癌中的功能，本研究构建了经短发夹RNA（shRNA）敲低FN1表达的K1细胞系，并筛选获得稳定敲低的单克隆细胞株；随后通过慢病毒（lentivirus）感染分别构建了EDB(+)FN1过表达与EDB(-)FN1过表达细胞株。