We’ve previously shown that microRNAs (miRNAs) miR-760 miR-186 miR-337-3p and miR-216b stimulate premature senescence through proteins kinase CK2 (CK2) down-regulation in individual colon cancer cells. USA). miRNA real-time quantitative PCR (qPCR) was performed using a TaqMan miRNA reverse transcription (RT) kit and by miRNA assay according to the manufacturer’s instructions with ABI PRISM 7000 HT (Applied Biosystems USA). The U48 small nucleolar RNA (RNU48) was used as the housekeeping small RNA reference gene. Real-time PCRs were run in triplicate for three different cDNAs. SA-β-gal activity assay SA-β-gal activity was measured as described previously (Dimri et al. 1995 with minor modifications. Cells in subconfluent cultures were washed with PBS fixed in 3% (v/v) formaldehyde in PBS for 10 min at room temperature and then incubated with a stain solution made up of 1 mg/ml of 5-bromo-4-chloro-3-indolyl-β-d-galactoside 40 mM citric acid-sodium phosphate (pH 6.0) 5 mM potassium ferricyanide 5 mM potassium ferrocyanide 150 mM NaCl and 2 mM MgCl2 for 24 h at 37°C. Blue-stained cells were counted in at least 10 areas at 400× magnification as well as the matters were expressed as the percentage of positive cells. Western blotting Cells in 60-mm dishes were washed with ice-cold PBS collected Poliumoside by scraping with a rubber policeman and lysed in 100 μl of ice-cold RIPA buffer [50 mM Tris-HCl (pH 8.0) 150 mM NaCl 1 NP-40 0.5% sodium deoxycholate 0.1% SDS 0.5 mM PMSF 1 μg/ml of aprotinin 1 μg/ml of leupeptin 1 μg/ml of pepstatin]. Western blotting was performed as described previously (Lee et al. 2013 Antibodies specific to CK2α p53 Poliumoside p21Cip1/WAF1 and β-actin were obtained from Santa Cruz Biotechnology (USA) and anti-HA antibody was obtained Poliumoside from Roche (Switzerland). Anti-p53 Poliumoside phospho-serine 392 antibody was from Cell Signaling Technology (USA). RT-PCR Total RNA was extracted from HCT116 cells. RNA was reverse-transcribed using gene-specific reverse primers and reverse transcriptase (Takara Japan) and the resulting cDNAs were PCR-amplified. PCR primer sequences for CK2α were CK2αFwd (5′-GACAAGCTTATGTCGGGACCC-3′) and CK2α Rev (5′-GACAAGCTTTTACTGCTGAGC-3′). The PCR primer sequences used for p53 were p53Fwd (5′-CCTCACCATCA-TCACACTGG-3′) and p53Rev (5′-CCTCATTCAGCTCTCGG-AAC-3′). The PCR primer sequences used for p21Cip1/WAF1 were p21Fwd (5′-GTGAGCGATGGAACTTCGACT-3′) and p21Rev (5′-CGAGGCACAAGGGTACAAGAC-3′). Primers specific to β-actin RNA were used to standardize the amount Poliumoside of RNA in each sample. PCR products were resolved on 1.5% agarose gel. Quantification of RT-PCR bands was performed using densitometry. Generation of mutant luciferase constructs and luciferase assay Human luciferase activities were measured consecutively using Dual Luciferase Assay (Promega Korea). Measurement of intracellular ROS Intercellular ROS level was decided using oxidation-sensitive fluorescent probes CM-H2DCFDA and dihydroethidium (DHE) as described previously (Jeon et al. 2010 Statistical analysis Statistical significance of the data was analyzed by one-way ANOVA with SPSS package program (SPSS Inc. USA). The results were considered significant if the value was less than 0.05. Duncan’s multiple-range test Poliumoside was also performed to test if the differences between the groups were identified at α = 0.05. RESULTS miR-760 and miR-186 are upregulated during replicative senescence in lung fibroblast IMR-90 cells Previously we exhibited that mimics of miR-760 PRKD2 miR-186 miR-337-3p and miR-216b together downregulated CK2α expression and prompted premature senescence in human colon cancer cells (Kim et al. 2012 To determine how the expression patterns of these miRNAs are affected by replicative senescence we repeatedly exceeded lung fibroblast IMR-90 cells until a senescence-like state was observed. Most cells at PDL 55 stained positive for SA-β-gal whereas only a few stained positive for SA-β-gal in early passage (PDL 33) cells (Fig. 1A). Western blot analysis revealed that the level of CK2α protein decreased in senescent cells (Fig. 1B) which corroborates previous results (Ryu et al. 2006 The protein amounts of p53 and p21Cip1/WAF1 increased in senescent cells. We validated the four miRNAs in cells using real-time qPCR. In comparison with proliferating IMR-90 cells (PDL33) miR-760 and miR-186 in senescent IMR-90 cells (PDL 55) increased by 180% and 240% respectively (Fig. 1C)..