Overexpression of miR-22 induced cellular senescence, cell cycle G1 arrest, and the decrease in BrdU incorporation in SiHa cells. (A and B) SiHa cells were transfected with cont miR or miR-22 at the indicated concentration for 6 d. qRT-PCR results show the relative level of miR-22 expression to cont miR in each transfection group (A). SA-β-gal activity was presented by the percentage of SA-β-gal–positive cells (B). (C) Cell morphology and SA-β-gal activity were analyzed by phase-contrast microscopy at day 6 after transfection of 10 nM miR-22 or cont miR. (D) Cell proliferation assay was performed after transfection of 10 nM miR-22, and cells were counted for the indicated days, compared with control cells. Each value was determined in triplicate. **, P < 0.01. (E) Cell cycle analysis was performed at 48 h after transfection. The percentage of G1, S, and G2 are demonstrated as shown. The histogram displays the relative changes of G1 and G2 phase compared with S phase. (F) BrdU quantitative analysis was performed at 72 h after transfection, presented by the percentage of BrdU incorporation. (G) Stable expression of miR-22 (Lenti-Pre22) was evaluated by qRT-PCR analysis, presented by the relative quantitation of miR-22 expression level at day 6 after infection. Expression level of miR-22 in Lenti-Pre22–transfected cells was relative to that in Lenti-C–transfected cells set at 1. U6 was used as an internal normalization control. (H) Cell morphology and SA-β-gal activity were analyzed by phase-contrast microscopy at day 6 after infection. The percentage of SA-β-gal–positive cells is presented in the right histogram. (I) Cell proliferation assay was performed at day 6 after infection 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.