Fig 2: Correlations between MBP-1, HDAC1 and ErbB2 protein expression in primary breast tumors. Correlation plot for MBP-1 versus HDAC1 (A), MBP-1 versus ErbB2 (B) and HDAC1 versus ErbB2 (C) protein levels. Black squares and coloured triangles represent expression values determined by immunohistochemical staining of 45 breast IDCs, as described in Materials and Methods. Blue lines represent the linear regression, dotted lines the 95% CI. The coefficient of correlation (r) was determined and its statistical significance was tested using the nonparametric Spearman rank correlation test.

Fig 3: scFv-9R/HER2si suppressed HER2 expression in EGFR-positive cells and inhibited cell proliferation and colony formationA and B. Lung cancer cells were either incubated with BSA/HER2si, scFv/HER2si or scFv-9R/HER2si, or transfected with HER2si using Lipofectamine. HER2 expression was determined by qRT-PCR (A) and Western blot (B), ß-action was used as internal control. C, D and E. Cells were cultured in the presence of BSA/HER2si, scFv/HER2si or scFv-9R/HER2si, and then subjected to MTT assay (C), colony-formation assay (D) and FCM analysis (E). A representative result was shown from 3 independent experiments. **p < 0.01.

Fig 4: DDX5 gene is co-amplified with MIR21 and DDX5 facilitates maturation of pri-miR-21. a Immunoprecipitation (IP) and western blotting analyses were performed using indicated antibodies. Normal immunoglobulin G (IgG) was used as a negative control for IP. The RNA-binding protein DDX1 was used as a positive control for the Drosha-binding proteins. b The DDX5-bound pri-miRNAs were immunoprecipitated with DDX5 and analyzed by qRT-PCR. Control IgG was used as a negative control. c Levels of primary or mature forms of miR-21 were analyzed in control and DDX5-knockdown breast cancer cells harboring 17q23 amplicon. d Levels of mature miR-21 and DDX5 were analyzed in breast tumor samples using in situ hybridization and immunohistochemistry. Representative staining images of tissue samples are shown. Scale bar, 100 µm. e Soft agar colony formation assays with MMTV-ErbB2 mouse mammary epithelial cells transduced with control vector or lentiviral vector expressing the indicated genes. Representative images are shown in the middle panel and relative expression levels of mature miR-16 or miR-21 are shown in the right panel. *p < 0.05; **p < 0.01; ***p < 0.001; unpaired 2-tailed t-test (b, c and e). Data are presented as mean ± SD and are representative of 3 independent experiments

Fig 5: Overexpression of miR-21 and WIP1 promotes oncogenic transformation of HER2+ breast cancer cells. a The differential expression heat map of miR-21 target genes in the mammary epithelial cells (MMECs) derived from MIR21-/-;MMTV-ErbB2 mice (8 weeks old). b Relative expression levels of the predicted miR-21 targets in primary MMECs isolated from wild-type or MIR21-/- virgin females at the age of 8 weeks. Data represents the mean expression levels normalized to the endogenous snoRNA55 control from three independent experiments. c MMECs derived from MMTV-ErbB2 mice were transduced with lentivirus expressing miR-21 and/or WIP1. Top panel: representative photomicrographs of SA-ß-galactosidase (SA-ß-Gal) staining observed in bright field. Bottom panel: miR-21 and WIP1 expression levels as determined by q-PCR, and percentages of SA-ß-Gal-positive cells were calculated. Scale bar, 10 µm. *p < 0.05; **p < 0.01; unpaired 2-tailed t-test was used (b, c). Data are presented as mean ± SD and are representative of 3 independent experiments