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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 effect of micro RNA-375 (mi R-375) on the expression of human epidermal growth factor receptor 2 (EMT) through the regulation of epithelial-mesenchymal transition (EMT) HER2) positive breast cancer cells to trastuzumab and its possible molecular mechanism. Methods: The sensitivity of trastuzumab to HER2-positive breast cancer cell line SKBR-3 and SK-BR-3R cell lines was detected by MTT assay and compared with real-time fluorescent quantitative PCR and Western blot Mi R-375 in cells and the expression levels of EMT-related proteins E-cadherin and vimentin. After the mi R-375 mimics were transfected into SK-BR-3R cells, the expression of mi R-375, E-cadherin and vimentin were detected by real-time fluorescence quantitative PCR and Western blotting. Sensitivity changes of drug cells to trastuzumab. Bioinformatics software was used to predict the mi R-375 target gene, and metadherin (MTDH) gene was selected as the target gene. The luciferase reporter gene system was further used to detect the transcriptional regulation of MTDH gene by mi R-375. The si RNA specific to MTDH gene was designed and transfected into SK-BR-3R cells. The expression of E-cadherin and vimentin in MTDH cells was detected by Western blotting. Results: The sensitivity of SK-BR-3R cells to trastuzumab was significantly lower than that of SK-BR-3 cells (P <0.001) The expression of mi R-375 and vimentin were significantly downregulated (P <0.001, P <0.05), while the expression of EMT characteristic protein E-cadherin was significantly increased (P <0.05). The expression of mi R-375 was significantly increased (P <0.001), the expression of vimentin was up-regulated (P <0.01) and the expression of E-cadherin was down-regulated in mi R-375 mimics transfected drug-resistant cells 0.001), and the sensitivity of drug-resistant cell lines to trastuzumab increased (P <0.001). mi R-375 negatively regulates MTDH gene transcription (P <0.001). After MTDH si RNA transfection, the expression of MTDH and E-cadherin were both down-regulated in drug-resistant cells (P <0.001), while the expression of vimentin was up-regulated (P <0.001). Conclusion: mi R-375 is involved in the resistance of trastuzumab to HER2-positive breast cancer cells by regulating EMT. This effect may be related to the regulation of MTDH expression.