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

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

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YAP13’-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=-0.5868,P=0.0028)。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。

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 SKBR-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=-0.5868,P=0.0028).After miR-375 mimic and YAP1-MUT cotransfection 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.