Fig. 3: MiR-133b overexpression inhibits tumorigenesis and metastasis in vivo.

a The tumor incidence of MDA-MB-231-luc cells with or without miR-133b overexpression. Cells were orthotopic injected into the mammary fat pad of mice at the indicated numbers. The tumor incidence was determined 3 months post injection. b-e In vivo tumorigenesis assays of MDA-MB-231-luc cells with or without miR-133b overexpression. In all, 5 × 10⁵ MDA-MB-231-luc-miR-133b or control cells were injected into the mammary fat pad of mice. b Left panel: Tumor volumes of MDA-MB-231-luc xenograft tumors infected with either control or miR-133b expressing vector at different days (n = 7 per group). Right panel: relative tumor volume at day 30 after injection of control or miR-133b expressing vector in nude mice. c Tumors were separated from the indicated mice and weighted after 30 days post injection. d The representative IHC staining images and the percentage of positive Ki67 cells in MDA-MB-231-luc cells-originated tumors at day 30 after injection. Red arrows point to Ki67-positive cells. e H&E stain of tissue adjacent to the indicated MDA-MB-231-luc primary tumors 30 days post injection. Arrows: disseminated tumor cells in normal fat (a, b) and muscle (c, d). f-h In vivo metastasis assays of MDA-MB-231-luc cells with or without miR-133b overexpression. Lung metastases were assessed by BLI measurements. The representative BLI images of mice at 4 week after injection are presented at left panel and quantitation of pulmonary metastases are show at right panel (n = 5 per group) (f). Wet lung weights in tumor-bearing mice were measured (g). Representative H&E staining of lungs shows metastatic nodules at ×40 and ×200 magnifications. Visible micrometastases were counted and analyzed (h). Data are presented as mean ± S.D. The symbol * denotes statistical difference (p < 0.05), whereas ** and *** indicate great significant difference (p < 0.01 and p < 0.001) following a two-tailed Student’s t-test. The scale bar represents 100 μm