Overexpression of miR-22 induces senescence-like phenotypes in human breast epithelial and breast cancer cells. (A) qRT-PCR analysis shows relative quantitation of miR-22 expression level (vs. 184hTERT) in human epithelial and various cancer cells. Expression levels of miR-22 in various cells were relative to that in 184hTERT cells set at 1. U6 was used as an internal normalization control. The dashed line represents the threshold of expression level (0.5-fold vs. 184hTERT). (B) Cell proliferation assay was performed after transfection of miR-22, and cells were counted for the indicated days, compared with control cells. Each value was determined in triplicate. *, P < 0.05. (C–E) Cell morphology and SA-β-gal activity were analyzed by phase-contrast microscopy at day 6 after transfection (C and D) or infection (E) in indicated cells. SA-β-gal activity was presented by the percentage of SA-β-gal–positive cells. (F) Cell proliferation assay was performed at day 6 after MDA-D3 cells were infected with Lenti-Pre22 and compared with control cells. Data in all the panels represent mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01. Bars, 50 µm.