Figure 2.

Overexpression of miR-411 Suppressed Anchorage-Independent Growth and Monolayer Proliferation and Induced Cell Cycle Arrest in G2/M Phase in Human BC Cells

(A and E) miR-411 and control vector plasmids were stably transfected into T24 (A) and UMUC3 (E) cells, and stable transfectants were identified by real-time PCR. Bars represent the mean ± SD, Student’s t test was used to determine the p value, and the asterisk (*) indicates a significant increase relative to control vector cells (*p < 0.05). (B and F) A soft-agar assay was performed to determine the effect of miR-411 overexpression on T24 (B) and UMUC3 (F) anchorage-independent growth. Representative images of colonies in the indicated cells were captured by microscopy after 3 weeks of incubation. (C and G) Colonies with >32 cells in T24 (vector) versus T24 (miR-411) cells (C) and UMUC3 (vector) versus UMUC3 (miR-411) (G) were counted, and the results are presented as colonies per 10,000 cells from three independent experiments. The asterisk (*) indicates a significant decrease relative to the vector control cells (p < 0.05). (D and H) The effect of miR-411 on the monolayer proliferative rates of T24 (D) and UMUC3 (H) cells were evaluated by ATP assays using a CellTiter-Glo Luminescent Cell Viability Assay kit. The results are presented as the mean ± SD, and the asterisk (*) indicates a significant inhibition of proliferation relative to vector control cells (p < 0.05). (I and J) The indicated cells were seeded into 6-well plates and cultured to 70%–80% confluence; after synchronization in 0.1% FBS medium for 24 hr, cells were cultured in complete medium for another 24 hr and then subjected to cell cycle analysis by flow cytometry as described in Materials and Methods, and G2/M arrest was induced by miR-411 in T24 (I) and UMUC3 (J) cells.