miR-375 modulates trastuzumab resistance in breast cancer cells. Trastuzumab-resistant SKBr-3 cells were infected with lentiviral vectors expressing pre-miR-control or pre-miR-375, and parental cells were transfected with a control RNA or a miR-375-specific inhibitor. A. qRT-PCR analyses of miR-375 expression. Data were normalized to mock-transfected cells and the expression levels of miR-375 were normalized to U6. B. MTT assays of modified cells after treatment with increasing concentrations of trastuzumab. C. Plate colony formation assays of modified trastuzumab-resistant cells in the presence of trastuzumab (5 μg/ml). D. Flow cytometry analyses of modified cells. Resistant and parental cells were treated with 10 μg/ml or 5 μg/ml trastuzumab, respectively, for 24 h, and then stained with FITC-conjugated Annexin V and PI. E. Microscopy images of modified trastuzumab-resistant SKBr-3 cells. Cells were treated with the indicated concentrations of trastuzumab for 24 h and images were captured using a phase contrast microscope. The arrow indicates autophagosome-like bodies. F. Upper panel: qRT-PCR assays were performed to identify the expression levels of miR-375 in human breast cancer BT474 and MDA-MB-453 cells. Lower panel: MTT assay of cells transfected with control or miR-375 antisense RNA and treated with various concentrations of trastuzumab for 24 h. G. Flow cytometry analyses of apoptosis in cells transfected with control or miR-375 antisense RNA. Twenty-four hours post-transfection, cells were treated with trastuzumab (5 μg/mL) for another 24 h, and cells were stained with FITC-conjugated Annexin V and PI. All data are represented as the mean ± SD or are representative of n = 3 replicates. *P <0.05, **P <0.01 and ***P <0.001.