Epigenetic mechanisms underlie miR-375 deregulation, and PI3K/Akt pathway is involved in miR-375/IGF1R-mediated trastuzumab resistance. A. The luciferase reporter construct of miR-375 promoter was introduced into parental and trastuzumab-resistant SKBr-3 cells, followed by assays of relative cellular luciferase activity. Data were normalized to the luciferase activity of control (vehicle transfected) cells. B. Cells were treated with 5-Aza-CdR (5 μmol/L) for 3 days and/or TSA (100 μmol/L) for 24 hrs. qRT-PCR was performed to quantify miR-375. NC, non-treated control. C. The levels of acetylated histone H3K9 in the miR-375 promoter region were determined by chromatin immunoprecipitation assay in indicated cells. NAC, nonspecific antibody control. Ac-H3, acetylated H3. D. Methylation-specific PCR analysis of miR-375 promoter region in parental (P) and trastuzumab-resistant (R) SKBr-3 cells. M, methylated allele; U, unmethylated allele. E. Western blot analyses of trastuzumab-resistant SKBr-3 cells expressing pre-miR-375 or control pre-miRNA, and parental SKBr-3 cells transfected with miR-375 antisense RNA or control RNA. GAPDH was used as the loading control. Data are represented as the mean ± SD of n = 3 replicates for A and B. **P <0.01 and ***P <0.001.