m^(6)A去甲基化酶FTO在甲状腺癌细胞中的作用及机制研究

Involvement of m^(6)A demethylase FTO in thyroid cancer cells

目的:探讨N^(6)-甲基腺苷(m^(6)A)去甲基化酶脂肪质量和肥胖相关蛋白(FTO)对甲状腺癌细胞m^(6)A甲基化和增殖、转移、侵袭等生物学行为的影响,及其对MAPK-JNK信号通路的调控机制,为甲状腺癌的防治提供理论基础。方法:使用FTO敲低质粒分别转染人甲状腺乳头状癌细胞TPC-1、人甲状腺鳞癌细胞SW-579、人甲状腺未分化癌细胞8505C后,经m^(6)A甲基化定量检测试剂盒检测甲状腺癌细胞m^(6)A甲基化修饰水平;分别采用平板集落形成实验、CCK-8实验,划痕实验,Transwell实验检测甲状腺癌细胞的增殖、迁移及侵袭能力;Western blot法检测MAPK-JNK信号通路相关蛋白表达水平的变化。结果:与未敲低FTO的细胞系比较,FTO敲低可提高甲状腺癌TPC-1、SW-579和8505C细胞的m^(6)A甲基化修饰水平,并促进甲状腺癌细胞的增殖、侵袭和迁移能力(P<0.01)。Western blot实验结果显示,FTO敲低后甲状腺癌细胞中E-cadherin和β-catenin的表达水平降低,N-cadherin和vimentin的表达升高,MAPK信号通路中的C-JUN和MLK3表达明显上调(均为P<0.05)。结论:在甲状腺癌细胞中,FTO敲低后促进甲状腺癌细胞的增殖、迁移和侵袭能力,并激活了MAPK信号通路,从而影响甲状腺癌的发生发展。

OBJECTIVE:To explore the effect of m^(6)A demethylase FTO on m^(6)A methylation,proliferation,metastasis,invasion and other biological behaviors of thyroid cancer cells,and its involvement with the MAPK-JNK signaling pathway.METHODS:The FTO knockdown plasmid was transfected into human thyroid papillary carcinoma cells(TPC-1),thyroid squamous carcinoma cells(SW-579),and undifferentiated thyroid carcinoma cells(8505C).Expression of m^(6)A in the cancer cells were measured using a m^(6)A methylation quantification detection kit.Their proliferation,migration,and invasion abilities were evaluated using the colony formation,CCK-8,wound healing,and Transwell assays.Changes in expression levels of proteins in the MAPK-JNK signaling pathway were examined using Western blot analysis.RESULTS:FTO knockdown increased m^(6)A expression levels in the TPC-1,SW-579,and 8505C cancer cells,and promoted their proliferation,invasion,and migration abilities(compared to cells without FTO knockdown,P<0.01).Western blot analysis revealed that FTO knockdown led to decreased expression of E-cadherin andβ-catenin,increased expression of N-cadherin and vimentin,and upregulated expression of C-JUN and MLK3 in the MAPK signaling pathway(compared with cells without FTO knockdown,all P<0.05).CONCLUSION:FTO knockdown enhanced proliferation,migration,and invasion abilities of thyroid cancer cells in vitro,and activated the MAPK signaling pathway,thereby potentially influenced development of thyroid cancer.These findings provide insights into the role of FTO demethylase in m^(6)A methylation and in thyroid cancer progression.