中国癌症杂志 ›› 2021, Vol. 31 ›› Issue (1): 27-34.doi: 10.19401/j.cnki.1007-3639.2021.01.004

• 论著 • 上一篇    下一篇

miR-375靶向YAP1调控上皮-间质转化参与乳腺癌细胞曲妥珠单抗的耐药

叶星明 1 ,王 淋 1 ,贾 静 1 ,吴秀凤 2 ,陈 颖 1   

  1. 1. 福建省肿瘤医院,福建医科大学附属肿瘤医院中心实验室,福建 福州 350014 ;
    2. 福建省肿瘤医院,福建医科大学附属肿瘤医院乳腺外科,福建 福州 350014
  • 出版日期:2021-01-30 发布日期:2021-02-20
  • 通信作者: 陈 颖 E-mail: fjbccy@hotmail.com
  • 基金资助:
    福建省卫生健康委员会中青年骨干项目(2016-ZQN-15);福建省自然科学基金(2018J01264);福建省医学创新基金(2017-CXB-2);福建省科技创新联合资金项目(2017Y9076)。

miR-375 targeting YAP1 modulates trastuzumab resistance through epithelial-mesenchymal transition in breast cancer cells

YE Xingming 1 , WANG Lin 1 , JIA Jing 1 , WU Xiufeng 2 , CHEN Ying 1   

  1. 1. Central Laboratory, Fujian Cancer Hospital & Fujian Medical University Cancer Hospital, Fuzhou 350014, Fujian Province, China; 2. Breast Surgery, Fujian Cancer Hospital & Fujian Medical University Cancer Hospital, Fuzhou 350014, Fujian Province, China
  • Online:2021-01-30 Published:2021-02-20
  • Contact: CHEN Ying E-mail: fjbccy@hotmail.com

摘要: 背景与目的:曲妥珠单抗作为人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)常用靶向抗体药物,其耐药问题日益凸显。探讨miR-375靶向Yes相关蛋白1(Yes-associated protein 1,YAP1)介导上皮-间质转化(epithelial-mesenchymal transition,EMT)参与乳腺癌细胞曲妥珠单抗的耐药。方法:建立HER2阳性乳腺癌曲妥珠单抗耐药的细胞株,转染miR-375 mimic来上调其表达,采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)检测miR-375的表达情况,采用蛋白质印迹法(Western blot)检测其EMT标志蛋白波形蛋白(vimentin)、E-钙黏蛋白(E-cadherin)的表达变化情况。采用MTT实验和平板克隆实验检测其药物敏感性及增殖能力的变化。双荧光素酶报告基因实验验证miR-375与YAP1 3’-UTR的靶向关系,采用RTFQ-PCR检测临床水平上两者的相关性。对细胞株共转染miR-375 mimic和YAP1-MUT载体后,采用Western blot检测其EMT蛋白表达的恢复情况,采用MTT实验和平板克隆形成实验检测其药物敏感性和增殖能力的恢复情况。结果:与NC组比较,miR-375 mimic组的药物敏感性、平板克隆形成能力均下调(P均<0.01),其EMT标志蛋白vimentin、E-cadherin表达也发生逆转(P均<0.05)。双荧光素酶报告基因实验结果证实,YAP1是miR-375的靶基因,miR-375与YAP1在乳腺癌患者肿瘤组织中呈负相关(r=-0.586 8,P=0.002 8)。miR-375 mimic组同时过表达YAP1后可恢复其药物敏感性(P<0.01)、克隆形成能力(P<0.05)和EMT标志蛋白vimentin、E-cadherin表达情况(P均<0.05)。结论:miR-375通过靶向YAP1在曲妥珠单抗耐药细胞株中介导EMT,进而调控曲妥珠单抗耐药细胞株的药物敏感性。

关键词: miR-375, 曲妥珠单抗耐药, 乳腺癌, Yes相关蛋白1, 上皮-间质转化

Abstract: Background and purpose: As the targeted antibody of human epidermal growth factor receptor 2 (HER2), trastuzumab resistance gets more and more attention. This study aimed to explore the role of miR-375 in the occurrence of trastuzumab resistance in breast cancer cells through regulating epithelial-mesenchymal transition (EMT) by targeting Yes-associated protein 1 (YAP1). Methods: Trastuzumab-resistant breast cancer cell line was established. miR-375 mimic and recombinant plasmid YAP1 were transfected into SK-BR-3R cells. The sensitivities of each breast cancer cell line to trastuzumab were detected by MTT assay, and proliferation was detected by colony formation assay. Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blot were applied to detect mRNA and protein expressions of miR-375, vimentin, E-cadherin and YAP1. Dual-luciferase reporter gene assay was conducted to verify the role of miR-375 in regulation of YAP1 transcription. A total of 25 breast cancer tissues from patients were collected to detect correlation in clinical performance. Results: Compared with NC group, after transfection with miR-375 mimic, the sensitivity to trastuzumab (P<0.01) and capacity of colony formation (P<0.01) for SK- BR-3R (resistance) cell line were decreased significantly, and the expression of vimentin was significantly downregulated, whereas E-cadherin was significantly upregulated (all P<0.05). YAP1 as downstream target gene of miR-375 was observed (P<0.01). MiR- 375 was negatively correlated with YAP1 in breast cancer tissues (r=-0.586 8, P=0.002 8). After miR-375 mimic and YAP1-MUT co- transfection in SK-BR-3R, compared with the control, the sensitivity to trastuzumab (P<0.01), capacity of colony formation (P<0.05) and the expressions of vimentin and E-cadherin were restored (P<0.05). Conclusion: miR-375 takes part in trastuzumab resistance of breast cancer cells through regulating EMT, which is mediated by targeting YAP1.

Key words: miR-375, Trastuzumab resistance, Breast cancer, Yes-associated protein 1, Epithelial-mesenchymal transition