miR-375通过调控EMT参与HER2阳性乳腺癌细胞对曲妥珠单抗的耐药

miR-375 modulates trastuzumab-resistance through EMT in HER2-positive breast cancer cells

目的 :探讨微RNA-375(micro RNA-375,mi R-375)通过调控上皮-间质转化(epithelial-mesenchymal transition,EMT),参与人类表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)阳性乳腺癌细胞对曲妥珠单抗耐药的作用及其可能的分子机制。方法:应用MTT法检测并比较HER2阳性乳腺癌亲本敏感细胞株SKBR-3和耐药细胞株SK-BR-3R对曲妥珠单抗的敏感性,采用实时荧光定量PCR和蛋白质印迹法检测两种细胞中mi R-375以及EMT相关蛋白E-钙黏蛋白(E-cadherin)和波形蛋白(vimentin)的表达水平。将mi R-375模拟物转染耐药细胞SK-BR-3R后,采用实时荧光定量PCR和蛋白质印迹法检测细胞中mi R-375、E-cadherin和vimentin表达水平的改变,MTT法检测耐药细胞对曲妥珠单抗的敏感性变化。应用生物信息学软件预测mi R-375的靶基因,并选取异黏蛋白(metadherin,MTDH)基因作为靶基因。进一步应用荧光素酶报告基因系统检测mi R-375对MTDH基因的转录调控作用。设计特异性针对MTDH基因的si RNA,并将其转染耐药细胞SK-BR-3R后,采用蛋白质印迹法观察MTDH表达被干扰后细胞中E-cadherin和vimentin表达水平的改变。结果 :与敏感细胞株SK-BR-3相比,耐药细胞株SK-BR-3R对曲妥珠单抗的敏感性明显降低(P<0.001),而且耐药细胞SK-BR-3R中mi R-375和vimentin的表达水平均明显下调(P<0.001,P<0.05),而EMT特征蛋白E-cadherin的表达水平明显上调(P<0.05)。mi R-375模拟物转染耐药细胞后,mi R-375的表达水平明显升高(P<0.001),vimentin表达水平明显上调(P<0.01),而E-cadherin表达水平下调(P<0.001),并且耐药细胞株对曲妥珠单抗的敏感性升高(P<0.001)。mi R-375对MTDH基因转录具有负向调控作用(P<0.001)。MTDH si RNA转染后,耐药细胞中MTDH和E-cadherin表达水平均下调(P值均<0.001),而vimentin表达水平上调(P<0.001)。结论:mi R-375通过调控细胞EMT,参与了HER2阳性乳腺癌细胞对曲妥珠单抗的耐药;这一作用可能与靶向调控MTDH表达有关。

Objective： To investigate the role of microRNA-375 （miR-375） in the occurrence of trastuzumab-resistance in human epidermal growth factor receptor 2 （HER2）-positive breast cancer cells through regulating epithelial-mesenchymal transition （EMT）. Methods： The sensitivities of HER2-positive breast cancer cell lines SK-BR-3 （a parental sensitive cell line） and SK-BR-3R （a trastuzumab-resistant cell line） to trastuzumab were detected by MTT assay. The expression levels of miR-375 and EMT-associated proteins E-cadherin and vimentin in the two cell lines were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. After miR-375 mimic was transfected into SK-BR-3R cells, the changes of miR-375, E-cadherin and vimentin expression levels were detected by real-time fluorescent quantitative PCR and Western blotting, and the change of trastuzumab sensitivity of SK-BR-3R cells was detected by MTT method. The potential target genes of miR-375 were predicted by bioinformatics software, and metadherin （MTDH） was selected as one of target genes. Luciferase reporter assay was conducted to verify the role of miR-375 in regulation of MTDH gene transcription. In addition, the changes of E-cadherin and vimentin expression levels in SK-BR-3R cells after transfection with MTDH siRNA were detected by Western blotting. Results： Compared to the sensitive SK-BR-3 cells, the sensitivity of SK-BR-3R cells to trastuzumab decreased significantly （P 〈 0.001 ）, and the expression levels of miR-375 （P 〈 0.001 ） and vimentin （P 〈 0.05） were significantly down-regulated, but the level of EMT marker E-cadherin was significantly up-regulated （P 〈 0.05）. After transfection with miR-375 mimic, the expression levels of miR-375 （P 〈 0.001） and vimentin （P 〈 0.01） were up-regulated, while the level of E-cadherin was down-regulated （P 〈 O.001）; the trastuzumab sensitivity of SK-BR-3R cells was increased （P 〈 0.001）. A negative regulatory effect of miR-375 on the transcription of MTDH gene was observed （P 〈 0.001）. After MTDH-siRNA transfection, the expressions of MTDH and vimentin were down-regulated （both P 〈 0.001）, while the expression of E-cadherin was up-regulated （P 〈 0.001）. Conclusion： miR-375 plays a role in drug-resistance of HER2-positive breast cancer cells to trastuzumab through regulating EMT, which is related to the target regulation of MTDH gene expression.