MIR-375 modulates trastuzumab-resistance through EMT in HER2-positive breast cancer cells / 肿瘤

ABSTRACT

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 < 0.001); 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.