Afamin对分化型甲状腺癌失分化与碘-131摄取功能的影响及机制

Differentiated thyroid cancer(DTC) sometimes loses the ability to take up iodine-131 by dedifferentiation, creating a major obstacle for radioiodine treatment to which there is currently no solution.In our preliminary work, we found that the low expression of afamin identified by differential proteomics analysis might be related to the dedifferentiation process and the loss of iodine-131 uptake capacity of DTC. To further explore the effect and mechanism of afamin on the dedifferentiation and iodine-131 uptake of DTC, in this research, afamin gene is transfected to papillary thyroid carcinoma cells K1 and undifferentiated thyroid carcinoma cells 8505c to enhance afamin expression using pEGFP-N1 plasmid as carrier (pathologically, 8505c cells was considered to be undifferentiated thyroid carcinoma by the dedifferentiation changes from well differentiated thyroid carcinoma). The empty plasmid transfected cells and untransfected cells are used as control. After transfection, the following changes were investigated: 1.the changes of the expression of MAPK signal pathway related-proteins (ERK1/2，p38, JNK1/2/3，ERK5) are analyzed by western-blot; 2.the changes of iodine-handling protein(NIS,TSHR,TPO,Tg), thyroid transcription factor(TTF1,TTF2,PAX8) and GLUT1 are analyzed both by real-time RT-PCR at mRNA level and by western-blot at protein level; 3.the changes of iodine-131 and 18FDG uptake are analyzed at cellular level(in vitro) and tumor-bearing nude mice models(in vivo). This original research is expected to clarify the role of afamin on the dedifferentiation and iodine uptake of DTC, and discovery of new therapeutic targets to enhance iodine-131 uptake for radioiodine treatment. Therefore, this research has significant scientific value, great practical significance and potential application.

分化型甲状腺癌（DTC）失分化导致碘-131摄取功能丧失是目前临床面临的主要难题。本课题组通过蛋白质组学方法筛选与验证，首次发现afamin蛋白低表达可能与DTC失分化有关。为进一步揭示afamin对DTC失分化和碘-131摄取的影响及机制，本研究以pEGFP-N1质粒为载体，将afamin基因转染至乳头状甲状腺癌细胞K1和由DTC失分化而来的未分化甲状腺细胞8505c中，以上调afamin的表达（以空质粒转染细胞和未转染细胞为对照）；然后检测细胞MAPK信号通路相关蛋白表达的变化，并在基因水平和蛋白水平检测NIS、TSHR、TPO、Tg和甲状腺转录因子及GLUT1表达的变化，最后在体外细胞水平和荷瘤裸鼠模型上检测K1和8505c细胞对碘-131和18FDG摄取的变化，以阐明afamin对DTC失分化的影响及机制，并探索发现提高碘-131摄取功能的新靶点。该研究具有重要科学价值和现实意义。

分化型甲状腺癌（Differentiated thyroid cancer，DTC）是内分泌系统最常见的恶性肿瘤，且其发病率在全球范围内逐渐升高。DTC转移灶对18F-FDG的摄取情况对DTC患者的预后具有重要的价值，即：转移灶具有高18F-FDG摄取预示相对不佳的预后。文献报道Afamin是某些肿瘤的标志物，但其在甲状腺癌细胞及分子水平的功能尚未被阐明。本研究使用人乳头状甲状腺癌（PTC）细胞株K1，未分化型甲状腺癌细胞株8505c及相应荷瘤裸鼠模型作为研究对象，对Afamin在甲状腺癌碘糖代谢中的作用做了系列研究。我们使用慢病毒转染技术成功构建了稳定表达Afamin的细胞株K1+AFM及8505c+AFM， 用实时荧光定量PCR及western blot来检测细胞基因表达变化。并用小动物PET/CT显像用来检测裸鼠皮下移植瘤对18F-FDG的摄取。本研究结果显示Afamin可能通过激活AKT/mTOR通路上调葡萄糖代谢相关蛋白（GAPDH, GLUT1, HK2）的表达，从而提高PTC细胞的葡萄糖代谢。体外18F-FDG摄取实验及体内小动物显像均表明PTC在稳定表达Afamin后，其葡萄糖代谢水平升高。另外，我们还发现稳定表达Afamin之后，PTC细胞中DNA损伤修复蛋白PARP1的表达水平显著升高，提示Afamin可能通过提高PARP1的表达增加分化型甲状腺癌细胞对碘-131的辐射耐受。本研究首次在细胞及分子水平研究了Afamin对甲状腺癌的影响，研究结果可为分化型甲状腺癌特别是难治性分化型甲状腺癌的诊治提供了新的思路；同时本研究结果首次报道Afamin对肿瘤细胞葡萄糖代谢产生的影响，阐明了该基因在肿瘤中可能的功能，为将来后续的研究提供了新的思路。此外，我们发现二甲双胍在体外和体内实验中均能通过下调HK2和GLUT1的表达水平从而降低DTC的糖代谢水平并抑制DTC细胞的增殖活性，该发现为碘难治性甲状腺癌的治疗提供了新的策略。