EGFR外显子19缺失突变及其在恶性甲状腺癌发生发展中的作用

EGFR exon 19 deletion reveals its role in malignant thyroid tumorigenesis and development

目的:研究表皮生长因子受体(epidermal growth factor receptor,EGFR)外显子19缺失突变在恶性甲状腺癌发生发展中的作用及其分子调控机制.方法:构建稳定转染EGFR外显子19缺失突变的正常人甲状腺滤泡上皮细胞(human thyroid follicular epithelial cell,HTFEC)株;EGF处理,通过划痕、Transwell、软琼脂克隆集落形成实验、失巢凋亡实验检测细胞的恶性转化能力;转录组测序分析(RNA-Seq),比较空载对照HTFEC细胞株和具有EGFR外显子19缺失突变的细胞株中显著差异表达的基因.结果:对50例多种类型的(恶性)甲状腺癌患者的二代测序分析显示,2例患者(4%)具有EGFR突变,其中一例为EGFR外显子19缺失突变的甲状腺癌来源肺腺癌(滤泡型),且第一代EGFR抑制剂(Icotinib)治疗极大地延缓了其肿瘤进展.实验结果表明,EGFR外显子19缺失突变能以EGF依赖性的方式转化HTFEC,使其具有侵袭特性.RNA-Seq分析显示,EGFR外显子19缺失突变后,LAMC2、TRIB3等基因表达显著上调,CTSB、FASN等基因表达显著下调.结论:EGFR外显子19缺失突变通过上下调与甲状腺功能密切相关的基因表达,如LAMC2、CTSB等,使HTFEC发生恶性转化,从而在甲状腺癌发生发展中发挥重要作用.靶向EGFR外显子19缺失突变可成为临床治疗恶性甲状腺癌的有效选择.

Objective:To investigate the effects and molecular regulation mechanisms of epidermal growth factor receptor(EGFR)exon 19 deletion on the occurrence and development of malignant thyroid cancer.Methods:We utilized normal human thyroid follicular epithelial cell(HTFEC)line to generate stable transfection cell line(HTFEC-ex19 del).The malignant transformation was examined by wound-healing,Transwell,soft agar colony formation and anoikis assay with EGF treated or not.The significant changes in mRNA levels of differentially expressed genes between HTFEC and HTFEC-ex19 del cells were observed by RNA-Seq analysis.Results:In our study,we found two cases with EGFR mutation in 50 thyroid cancer patients by next-generation sequencing,one of which was a thyroid-derived lung cancer patient(follicular type)harbored EGFR exon 19 deletion and responded to the first generation of EGFR inhibitor(Icotinib).The result showed that EGFR exon 19 deletion could transform HTFEC in an EGF dependent manner to make it invasive.Data of RNA-Seq from generated two cell lines(control HTFEC and HTFEC-ex19 del)uncovered that the expression of LAMC2,TRIB3 and other genes was significantly up-regulated,while the expression of CTSB,FASN and other genes was significantly down-regulated in HTFEC-ex19 del cells.Conclusion:EGFR exon 19 deletion mutation plays a critical role in the occurrence and development of thyroid cancer by up-or down-regulating the expression of genes closely related to thyroid function,such as LAMC2,CTSB,etc.,which leads to the malignant transformation of HTFEC.Inhibitors targeting EGFR can be an effective therapy of malignant thyroid cancer.