粒状头样蛋白2下调诱导上皮间质转化促进肿瘤细胞对吉非替尼耐药  

Down-regulation of grainyhead-like protein 2 promotes drug resistance of tumor cell to gefitinib by inducing epithelial-mesenchymal transformation

作　　者：洪永刚[1] 郝立强[1] 毕峰瑞 颜宏利[2] HONG Yong-gang;HAO Li-qiang;BI Feng-rui;YAN Hong-li(Department of Colorectal Surgery,Changhai Hospital,Navy Medical University(Second Military Medical University),Shanghai 200433,China;Department of Reproductive Medicine,Changhai Hospital,Navy Medical University(Second Military Medical University),Shanghai 200433,China)

机构地区：[1]海军军医大学(第二军医大学)长海医院肛肠外科,上海200433 [2]海军军医大学(第二军医大学)长海医院生殖医学中心,上海200433

出　　处：《第二军医大学学报》2018年第10期1102-1108,共7页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(81472770)~~

摘　　要：目的探讨粒状头样蛋白2(GRHL2)在肿瘤靶向治疗药物表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)吉非替尼获得性耐药中的作用和可能机制。方法利用吉非替尼浓度逐步递增的体外诱导方法培养人结直肠癌细胞DiFi和人肺腺癌细胞HCC4006,获得吉非替尼耐药细胞株。通过RNA测序方法筛选在耐药细胞株与其亲代细胞中差异表达的基因并进行实时荧光定量PCR验证。使用pcDNA3.1-GRHL2质粒转染耐药细胞株使GRHL2过表达,用CCK-8法检测细胞对吉非替尼的敏感性。采用蛋白质印迹法检测耐药细胞株中上皮标志物E-cadherin和间质标志物Vimentin的表达变化,并通过CellMinerTM数据库分析60株人肿瘤细胞系中GRHL2表达与E-cadherin和Vimentin表达的关系。结果成功获得DiFi和HCC4006的耐药细胞株。通过RNA测序和实时荧光定量PCR验证发现GRHL2在耐药细胞株中表达下调,而在耐药细胞株中过表达GRHL2后细胞恢复了对吉非替尼的敏感性。蛋白质印迹分析表明耐药细胞株中上皮标志物E-cadherin表达下调,而间质标志物Vimentin表达上调;CellMinerTM数据库分析表明GRHL2的表达与E-cadherin/Vimentin比值高度一致。结论 GRHL2表达下调通过诱导上皮间质转化介导肿瘤细胞对吉非替尼的耐药。Objective To explore the role and possible mechanism of grainyhead-like protein 2(GRHL2)downregulation in acquired drug resistance to tumor-targeted therapeutic drug gefitinib,an epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI).Methods Human colon cancer cell line DiFi and human lung adenocarcinoma cell line HCC4006 were cultured in a stepwise increasing concentration of gefitinib to obtain gefitinib-resistant cell lines.The differentially expressed genes between gefitinib-resistant cell lines and parent cells were selected by RNA sequencing and verified by real-time fluorescent quantitative PCR(qRT-PCR).The pcDNA3.1-GRHL2 plasmid was transfected into the gefitinib-resistant cell lines to overexpress GRHL2,and the sensitivity of the cells to gefitinib was detected by CCK-8 method.The expression of epithelial marker(E-cadherin)and mesenchymal marker(Vimentin)in the gefitinib-resistant cells was detected by Western blotting.The relationship between GRHL2 expression and expression of E-cadherin and Vimentin in 60 human tumor cell lines was analyzed by CellMinerTM database.Results We successfully obtained two gefitinib-resistant cell lines.RNA sequencing and qRT-PCR confirmed that the expression of GRHL2 in the gefitinib-resistant cells was decreased,while the sensitivity of the cells to gefitinib was restored after overexpressing GRHL2 in the gefitinib-resistant cells.Western blotting analysis showed that the E-cadherin expression was decreased and the Vimentin expression was increased in the gefitinib-resistant cell line.CellMiner^TM database analysis showed that the expression of GRHL2 was highly consistent with the ratio of E-cadherin to Vimentin.Conclusion Down-regulation of GRHL2 mediates drug resistance of tumor cell to gefitinib by inducing epithelial-mesenchymal transition.

关 键 词：靶向治疗 继发性耐药 吉非替尼 粒状头样蛋白2 上皮间质转化 

分 类 号：R730.5[医药卫生—肿瘤]