天津医药

天津医药 ›› 2020, Vol. 48 ›› Issue (1): 8-13.doi: 10.11958/20192340

• 细胞与分子生物学 • 上一篇    下一篇

Wnt信号通路转录上调EGFR促进非小细胞肺癌 吉非替尼耐药

王倩，黎谢梦丹，罗凯，郑国沛，张志杰，卢敏莹，董静，贾小婷△，贺智敏   

  1. 基金项目：国家自然科学基金资助项目（81872450）；广州市卫生和计划生育科技项目（20181A011093，20191A011099）；广东省医学科学 技术研究基金项目（B2019063）；广州市医学重点学科建设项目 作者单位：广州医科大学附属肿瘤医院，广州医科大学肿瘤研究所（邮编510095） 作者简介：王倩（1980），女，硕士，副主任医师，主要从事肺、乳腺肿瘤的发生与靶向治疗耐药方面研究 △通讯作者 E-mail：jxt1231994@163.com
  • 收稿日期:2019-08-01 修回日期:2019-10-30 出版日期:2020-01-15 发布日期:2020-01-15
  • 通讯作者: 罗凯 E-mail:luokainan@126.com
  • 基金资助:
    中国国家自然科学基金;广东省医学科学技术研究基金项目;广州市卫生和计划生育科技项目;广州市医学重点学科建设项目;广州市卫生和计划生育科技项目

Transcriptional upregulation of EGFR by Wnt signaling pathway to promote gefitinib resistance in non-small cell lung cancer

WANG Qian, LI Xie-meng-dan, LUO Kai, ZHENG Guo-pei, ZHANG Zhi-jie, LU Min-ying, DONG Jing, JIA Xiao-ting△, HE Zhi-min   

  1. The Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou 510095, China △Corresponding Author E-mail: jxt1231994@163.com
  • Received:2019-08-01 Revised:2019-10-30 Published:2020-01-15 Online:2020-01-15

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摘要： 目的 探讨Wnt信号通路和表皮生长因子受体（EGFR）在非小细胞肺癌（NSCLC）吉非替尼耐药中的作用 及其机制。方法 运用实时荧光定量PCR（qRT-PCR）和免疫印迹实验（Western blot）检测EGFR在亲代HCC827细 胞与吉非替尼耐药细胞（HCC827/R）中的表达；免疫组化染色检测耐药前后3对NSCLC肿瘤组织中EGFR的表达。 双荧光素酶报告基因实验（Luciferase）检测亲代 HCC827 细胞与耐药 HCC827/R 细胞 Wnt 信号通路活化情况；在 Jaspar数据库中预测EGFR基因启动子区上TCF/LEF转录因子结合位点；染色质免疫共沉淀实验（Chip）和Luciferase 实验检测转录因子对基因表达的调控；功能阻断实验检测Wnt信号通路/EGFR途径介导吉非替尼耐药的作用。结 果 与亲代HCC827细胞相比较，HCC827/R细胞的EGFR在mRNA和蛋白水平表达均明显增高（P＜0.05）。免疫组 化染色结果显示在 3 例吉非替尼耐药前后 NSCLC 配对肿瘤组织样品中有 2 例耐药后 EGFR 表达较耐药前增加。 Luciferase实验结果显示，与亲代细胞比较，Wnt/β-catenin信号通路在耐药HCC827/R细胞中异常活化（P＜0.05）。生 物信息学分析预测到EGFR基因启动子区-1476~-1468区域存在Wnt/β-catenin信号通路下游转录因子TCF3/TCF4 结合位点，Chip和Luciferase实验证实Wnt/β-catenin信号通路可转录上调EGFR表达。功能阻断实验结果显示当利 用Wnt3a刺激亲代HCC827细胞的同时敲低EGFR，吉非替尼对细胞的抑制率较单独利用Wnt3a刺激细胞时的细胞 抑制率得到恢复（P＜0.05）。结论 Wnt/β-catenin信号通路转录上调EGFR促进NSCLC的吉非替尼耐药，其为提高 NSCLC吉非替尼靶向治疗效果提供了新的实验依据。

关键词: 癌, 非小细胞肺, Wnt信号通路, 受体, 表皮生长因子, 吉非替尼, 耐药性

Abstract: Objective To investigate the effect of Wnt signaling pathway and epidermal growth factor receptor (EGFR) on gefitinib resistance of non-small cell lung cancer (NSCLC) and its mechanism. Methods EGFR expressions in parental cells and gefitinib-resistant HCC827 / R cells were detected by qRT-PCR and Western blot assay. Immunohistochemical (IHC) staining was used to detect the expressions of EGFR in NSCLC tissues before and after drug resistance. Activation status of Wnt signaling pathway in cells were detected by luciferase assay. TCF/LEF transcription factor binding sites on the EGFR promoter region were predicted in the Jaspar database. The regulation of transcription factors on gene expression was detected by Chip and luciferase assays. Functional blockade assay was used to detect whether Wnt signaling pathway/EGFR pathway mediated gefitinib resistance. Results Compared with the parental cells, the expression of EGFR in HCC827/R cells was significantly increased at mRNA and protein levels (P＜0.05). The results of IHC showed that high expressions of EGFR in NSCLC tissue samples after drug resistance were detected in 2 of 3 paired NSCLC tissue samples before and after gefitinib resistance. Luciferase results showed that Wnt/β-catenin signaling pathway was abnormally activated in resistant HCC827/R cells compared with that of parental cells (P＜0.05). Bioinformatics analysis predicted that there were TCF3/ TCF4 sites, which were downstream transcription factors of the Wnt/β-catenin signaling pathway, the binding site located on the promoter region of EGFR gene (- 1476~ - 1468), and Chip and Luciferase experiments confirmed that EGFR gene expression could be up-regulated by Wnt/β - catenin signaling pathway (P＜0.05). The results of functional blockade experiments showed that when Wnt3a was used to stimulate parental HCC827 cells while knocking down EGFR, the inhibition rate of 3.13 μM gefitinib on cells was restored compared with that of Wnt3a alone (P＜0.05). Conclusion Wnt/ β-catenin signaling pathway up-regulates EGFR expression to promote gefitinib resistance in NSCLC, which provides the new experimental evidence for improving the therapeutic effect of gefitinib targeted therapy on NSCLC.

Key words: carcinoma, non-small-cell lung, Wnt signaling pathway, receptor, epidermal growth factor, gefitinib, drug resistance