L cell line HT29 to varying durations of hypoxia and saw a similar, albeit less

L cell line HT29 to varying durations of hypoxia and saw a similar, albeit less striking, reduction in Dicer mRNA levels (Figure 2B). However, no significant changes in Dicer mRNA levels were observed in SKBR3 breastcancer cells after being exposed to varying durations of hypoxia (24 h and 48 h, at 0.1 O2) despite seeing substantial reductions in Dicer protein expression.The mechanism of hypoxic repression of Dicer The effect of HIF hydroxylase inhibitors on Dicer levelsHIF plays a central role in the Vesatolimod web transcriptional response to hypoxia, so the role of the HIF pathway in the hypoxic repression of Dicer levels was examined. Cells were exposed to the HIF hydroxylase inhibitors, dimethyloxalyl glycine (DMOG) and desferrioxamine which induce HIF levels under normoxic conditions [36,39]. A modest decrease in Dicer mRNA levels was seen after exposing MCF7 cells to DMOG (1 mM) for 48 h (Figure 3A) and desferrioxamine (0.1 mM) for 48 h (Figure 3B). A similar influence on Dicer protein levels was seen followingFigure 2 Dicer mRNA expression in hypoxia vs. normoxia. Dicer mRNA expression was examined in MCF7 and HT29 cells after exposure to 0.1 O2 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/26780312 for different durations. A, Dicer mRNA expression in MCF7 cells after exposure to 0.1 O2 for 8 h (P = 0.0007), 16 h (P = 0.0009), 24 h (P = 0.02) and 48 h (P = 0.008). B, Dicer mRNA expression in HT29 cells after exposure to 0.1 O2 for 16 h (P = 0.01), 24 h (P = 0.02) and 48 h (P = 0.09). *denotes P < 0.05 compared with parallel controls in normoxia. Data represent normalized mean ?S.E (error bars) (n = 3). Dicer mRNA levels were analysed by RT-PCR and normalised to 18S rRNA levels. Statistical significance established by Student's t-test.Bandara et al. BMC Cancer 2014, 14:533 http://www.biomedcentral.com/1471-2407/14/Page 6 ofFigure 3 Dicer mRNA and protein expression after exposure to HIF hydroxylase inhibitors. Dicer mRNA and protein expression was examined in MCF7 and SKBR3 cells after exposure to HIF hydroxylase inhibitors: dimethyloxalyl glycine (DMOG) and desferrioxamine (DFO) A, Dicer mRNA expression (P = 0.04) in MCF7 cells after exposure to 1 mM DMOG for 48 h. B, Dicer mRNA expression in SKBR3 cells after exposure to 0.1 mM DFO for 48 h. *denotes P < 0.05 compared with parallel controls. Data represent normalized mean ?S.E (error bars) (n = 3). Dicer mRNA levels were analysed by RT-PCR and normalised to 18S rRNA levels. Statistical significance established by Student's t-test. C, Dicer protein expression in MCF7 cells after exposure to 1 mM DMOG for 48 h Results show two technical replicates per treatment. D, Dicer protein expression in SKBR3 cells after exposure to DFO (0.1 mM) for 48 h. Results show three technical replicates per treatment. Dicer and -actinin protein expression was examined by immunoblotting. -actinin was used as the loading control.exposure to HIF hydroxylase inhibitors. A modest decrease in Dicer protein levels was seen in MCF7 cells after exposure to DMOG (1 mM) for 48 h (Figure 3C). A more substantial decrease in Dicer protein levels were seen in SKBR3 cells after exposure to desferrioxamine (0.1 mM) for 48 h (Figure 3D).Involvement of HIF in Dicer regulationinhibited simultaneously using RNA interference there was no effect on the DICER mRNA repression, suggesting a lack of HIF-1 or HIF-2 mediated down regulation of DICER mRNA levels after exposure to hypoxia (Figure 4B). Substantial repression of Dicer protein was observed under hypoxia but there was no significant.