基于CRISPR/Cas9技术研究钠碘同向转运体在甲状腺癌放射性碘抵抗中的分子作用机制

Radioactive iodine (RAI) is an important strategy for the treatment of Differentiated Thyroid Carcinoma (DTC) patients, but some of the patients will be the occurrence of Radioactive Iodine Resistance (RAIR). In BRAF mutation thyroid carcinoma cells, MEK, ERK overexpressed, which affected the expression and localization of Sodium Iodide Symporter (NIS), but the mechanism is not clear. We found that the iodine uptaking decreased in BRAF mutation thyroid cancer cell line WRO in vitro and immunocytochemistry(ICC) showed that the NIS was dispersed in the cytoplasm. Iodine uptaking increased after the usage of U-O126 (MEK inhibitor) and NIS was located at the membrane. There existed close relationship between MEK and NIS through co-immunoprecipitation (Co-IP). Phos-tag detected that NIS phosphorylation degree was significantly higher in BRAF mutant cell lines than in BRAF wild-type cell lines. Therefore, we hypothesize that the BRAF mutation may decrease iodine uptaking through NIS overphosphorylation. We predict three phosphorylation sites, and will use Site-directed Gene Mutagenesis technique and mass spectrometry analysis to verify the phosphorylation sites, to find the NIS phosphorylation kinase. CRISPR/CAS9 was used to establish MEK and ERK gene knockout thyroid cancer cell line, iodine uptaking in vitro and small animal PET/CT and other means to demonstrate whether the BRAF mutation make NIS overphosphorylation through activation of downstream genes. Finally figure out the mechanism of MAPK signaling pathway to RAIR in the DTC patients and provide new treatment ideas for them.

碘同位素是治疗分化型甲状腺癌的重要手段，但有些患者会出现放射性碘抵抗（RAIR）现象。目前认为可能与BRAF突变影响钠碘同向转运体（NIS）的功能有关，但其作用机制尚不清楚。预实验发现BRAFmutant细胞株体外摄碘低且NIS在胞浆内弥散分布，采用MEK抑制剂后摄碘率增高，NIS可重新定位于细胞膜；免疫共沉淀技术揭示ERK与NIS两种蛋白关系密切；Phos-tag技术检测到BRAFmutant细胞株中NIS磷酸化明显高于BRAFwt细胞株。因此我们推测BRAFmutant突变可能通过MEK-ERK介导NIS过度磷酸化从而降低甲状腺癌摄碘功能。我们预测了三个磷酸化位点，并将采用定点突变和质谱分析等技术验证；通过CRISPR/CAS9技术建立基因敲除的甲状腺癌细胞系，采用细胞体外摄碘、小动物PET/CT等手段探索MAPK途径对甲状腺癌细胞摄碘下降的作用机制，为RAIR患者提供新的治疗思路。

背景：甲状腺乳头状癌（PTC）是一种MAPK驱动的肿瘤。MAPK信号通路激活已被证实与放射性碘（RAI）抵抗机制有关，但对小分子抑制剂的耐药性限制了其在RAI中的再分化中的作用。.目的和方法：本研究课题中，我们使用CRISPR/Cas9敲除BRAFV600E突变的K1和BCPAP细胞中MEK1/2基因，并同时使用雷帕霉素共同靶向MAPK和PI3K-AKT-mTOR通路，以探讨它们是否能在PTC中发挥协同抗肿瘤和再分化功能。然后，我们检测了与mTOR抑制剂雷帕霉素联合使用时的MEK1/2基因敲除甲状腺肿瘤细胞的生物学功能和体内外碘摄取情况。.结果：U-0126作为MEK的小分子抑制剂，能有效阻断MAPK通路，增加NIS的表达，促进PTC细胞对RAI的摄取。然而，随着时间的推移，其阻断效率下降。CRISPR/cas9敲除MEK1/2基因后，NIS上调，K1和BCPAP细胞对RAI的摄取增加。而MEK1/2基因敲除对细胞周期、凋亡和增殖的抑制作用无明显差异。同时使用mTOR抑制剂雷帕霉素抑制MEK1/2敲除细胞的增殖并促进细胞凋亡。此外，我们发现雷帕霉素促进NIS的糖基化，然后引导NIS蛋白在细胞膜上富集。.结论和意义：联合靶向抑制MEK和mTOR可能是治疗PTC患者对MEK抑制剂耐药性的潜在策略。