miR-22 is up-regulated in senescent human fibroblasts, mediating cellular senescence. (A) miRNA expression profile of TIG-3 fibroblasts was analyzed by miRNA microarray, presented as fold changes in miRNA expression between TIG-3S (senescent) and young (Y) cells. A set of altered expression miRNAs is indicated by red columns (see Table S1). RQ, relative quantitation. (B) Relative quantitation of miR-22 expression in different PDLs of fibroblasts was analyzed by qRT-PCR analysis. miR-22 expression levels in human fibroblasts were indicated, relative to those in TIG-3 (42 PDL) set at 1 in the left histogram and MRC-5 (43 PDL) set at 1 in the right histogram. U6 was used as an internal normalization control. The dashed line represents the threshold of expression level (twofold vs. TIG-3 42 PDL). (C and D) MRC-5 cells were transfected with cont miR or mature miR-22 (miR-22) for 6 d at indicated concentration. (C) qRT-PCR analysis shows the relative quantitation of miR-22 expression (vs. cont miR) in each transfection group. miR-22 expression levels in miR-22–transfected MRC-5 cells were indicated, relative to that in cont miR–transfected cells set at 1. U6 was used as an internal normalization control. (D) SA-β-gal activity was presented by the percentage of SA-β-gal–positive cells, which was indicated in different dose groups. (E) Cell proliferation assay was performed after transfection of 10 nM miR-22 or cont miR, and cells were counted for the indicated days. Each value was determined in triplicate. **, P < 0.01. (F) Representative photos for SAHF formation in MRC-5 cells at day 6 after transfection. Images were taken with fluorescence microscopy. Enlarged images of the boxed area from the top are shown in the bottom. SAHF formation was quantified by counting 200 cells from >10 random fields, and the results were shown in the right histogram in contrast to MRC5S (senescent; 58 PDL). Data in all the panels represent mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01. Bars, 20 µm.