Synthetic miR-22 delivery induces cellular senescence in vivo and inhibits breast tumor growth and metastasis in vivo. (A and B) Representative fat pad orthotopic tumor (A) and selected organ (B) images of mice on day 46 after inoculation. Quantitation of bioluminescence emitted from the primary tumor (A) or whole body (B) of mice was presented as the mean ± SEM (n = 6). L, liver; K, kidney; Sp, spleen; Si, small intestine; St, stomach. (C) Relative quantitation of miR-22 level in primary tumor tissues was analyzed by qRT-PCR. Total RNA was isolated from five 10-µm-thick optimal cutting temperature compound–embedded frozen tissue sections. Expression level of miR-22 in miR-22–treated tumors was relative to that in cont miR–treated tumors set at 1. U6 was used as an internal normalization control. Data represent mean ± SEM (n = 5). (D and E) Representative histochemical detection of SA-β-gal activity in vivo (D) and hematoxylin and eosin (HE) staining (E) in primary tumor on day 46 after inoculation. Images were taken with light microscopy. Seven independent fields were chosen, and the percentage of SA-β-gal–positive cells is presented in the histogram. Data represent mean ± SEM (n = 4). Bars, 50 µm. (F) Effect of miR-22 on cell proliferation in primary tumor tissues was evaluated by Ki-67 immunostaining. The histogram displays the percentage of Ki-67 labeling index in these groups. Data represent mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01.