Alteration in hypoxic repression of Dicer protein after HIF-1 RNA interference (Figure 4C).Prolyl hydroxylase dependent regulation of DicerTo further investigate the involvement of HIF in Dicer regulation RNA interference was used to inhibit HIF-1 and HIF-2, and Dicer levels was examined. The HIF-1 RNA interference substantially reduced the hypoxic expression of HIF-1 protein (Figure 4A). The influence of HIF-1 and HIF-2 isoforms in hypoxic repression of Dicer was examined. When HIF-1 and HIF-2 wereThese earlier results pointed towards a mechanism of regulation that was oxygen and HIF hydroxylase dependent but was independent of HIF-1. To investigate this we used siRNA mediated suppression of PHD2 expression in SKBR3 cells and determined the levels of Dicer. ABandara et al. BMC Cancer 2014, 14:533 http://www.biomedcentral.com/1471-2407/14/Page 7 ofFigure 4 Dicer repression in hypoxia is not HIF dependent. Dicer expression was examined after HIF-1 and HIF-2 inhibition with siRNA in hypoxia compared to normoxia A, HIF-1 protein expression in SKBR3 cells after transient transfection with a HIF-1 targeting siRNA or control siRNA, then exposure to hypoxia (0.1 O2 for 24 h) vs. normoxia. Results show two technical replicates per treatment. B, Dicer mRNA expression in MCF7 cells after transient transfection with HIF-1, HIF-2 or control siRNA, then exposure to hypoxia (0.1 O2 for 48 h) vs. normoxia. Data represent normalized mean ?S.E (error bars) (n = 3). Dicer mRNA levels were analysed by RT q-PCR and normalised to -2-microglobulin mRNA levels. C, Dicer protein expression in SKBR3 cells after transient transfection with HIF-1 targeting siRNA or control siRNA, then exposure to hypoxia (0.1 O2 for 48 h) vs normoxia. Results show two technical replicates per treatment. Dicer and -actinin protein levels were examined by immunoblotting. -actinin was used as the loading control.significant decrease in Dicer mRNA (P = 0.0008) (Figure 5A) and a large decrease in Dicer protein (Figure 5B) levels, were evident following PHD2 suppression in normoxia. This observation was validated by repeating the experiment with a different PHD2 siRNA and a similar effect was seen on Dicer expression. PHD2 suppression did not affect Drosha and TARBP2 protein levels (Figure 5C). We also examined if the other HIF hydroxylase enzymes (PHDs or Factor Inhibiting HIF-1 (FIH-1)) were involved in Dicer regulation by hypoxia. In contrast to the effects of PHD2 suppression, there was no decrease in Dicer expression after PHD1 (Figure 5D) or FIH-1 RNA interference (Figure 5E). In keeping with previous work, PHD3 levels were very low under normoxic buy Peficitinib conditions [40].The involvement of microRNAs in Dicer regulation by hypoxiaIn other experimental settings, the feedback regulation of Dicer by specific Dicer dependent miRNAs has been reported to PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27385778 be important in controlling Dicer levels [41,42]. We therefore hypothesised that a hypoxically induced miRNA might mediate the hypoxic repression of Dicer expression. miR-210 is the best characterised example of a miRNA that shows substantial induction under hypoxic conditions. We examined the influence of miR-210 manipulations on Dicer expression. miR-210 was over-expressed in normoxia by transient transfection of SKBR3 cells with a miR-210 mimic and a control mimic (Figure 6A). We determined Dicer mRNA (Figure 6B) and protein (Figure 6C)Bandara et al. BMC Cancer 2014, 14:533 http://www.biomedcentral.com/1471-2407/14/Page 8 ofFigure 5 Prol.
