These homologues genes may regulate beetle developmental processes by influencing hormone levels

either FPN promoter construct. Quercetin decreases 39-UTR reporter activity in Caco-2 cells Polyphenols are known to be potent regulators of micro-RNA expression and activity. We therefore investigated whether quercetin might regulate FPN mRNA levels in Caco-2 cells via miRNA interaction with its 39UTR. In reporter assays in Caco-2 cells transfected with a Renilla-luciferase reporter construct containing the 39 UTR of human FPN, exposure to quercetin significantly decreased reporter activity. Next we attempted to identify potential miRNA species that might be involved in these regulatory events. First, we used arrays to identify miRNA species up-regulated by quercetin in Caco-2 cells. Expression of 33 miRNAs were increased.1.5-fold in quercetin-treated cells compared with untreated controls. Next we used web-based bioinformatics packages to determine Quercetin did not alter FPN MedChemExpress STA 4783 59-promoter activity in Caco2 cells Next we investigated whether the inhibitory effects of quercetin on FPN protein and mRNA were mediate via altered gene transcription. Two FPN mRNA isoforms have been identified in Caco-2 cells; FPN1A PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19659763 and FPN1B, respectively. Reporter assays with luciferase reporter constructs containing 1Kb of either the FPN1A or FPN1B 59-promoter indicated that FPN1A is the 4 Quercetin and Intestinal Iron Absorption if any up-regulated miRNAs had consensus binding sites in the human FPN 39-UTR. Binding sites for 2 members of the miR-17 family, miR-17-3p and miR-17-5p, respectively, were present in the FPN 39UTR. Finally, we used qPCR to validate the array data to determine whether expression of the miR-17 species was altered following quercetin treatment. miR-17-3p was strongly up-regulated in quercetin-treated Caco-2 cells. In contrast, there was no significant effect of quercetin on the expression of miR-17-5p. Furthermore, expression levels of miR-485-3p, which has 2 consensus binding sites in the FPN 39UTR and has been shown to be involved in the inhibition of FPN expression by iron deficiency in HepG2 cells, were not altered in quercetin-treated Caco-2 cells. Control Duodenal non-haem iron Serum iron Transferrin saturation Liver hepcidin mRNA Data are means 6 SEM. P,0.05 compared with control group. doi:10.1371/journal.pone.0102900.t001 NS Quercetin-treated 32.561.5 90.763.9 18.961.0 0.260.1 NS 22.961.2 140.9615.1 27.662.5 0.660.3 Not significantly different from control group. 5 Quercetin and Intestinal Iron Absorption Discussion Several independent studies have established that polyphenols significantly decrease non-haem iron absorption in duodenum; however, the exact mechanism of action is unknown. Based on the data from our current study we discuss four possible mechanisms that might underlie the inhibitory effects of quercetin on intestinal iron transport: 1. Iron chelation; 2. Circulating iron and hepcidin levels; 3. Cellular iron levels; 4. Direct effects of quercetin on intestinal iron transporter expression. 6 Quercetin and Intestinal Iron Absorption 1. Iron chelation Our acute in vivo iron transport data demonstrated that quercetin increases iron uptake and retention by the duodenal mucosa. We argued that iron chelation by quercetin could play important role in this phenomenon. It has previously been shown that the preferred site for iron chelation by quercetin is between the 3-hydroxyl and 4-carbonyl group; for complexes containing one iron and one quercetin molecule, binding strength has an order 34.45.3949. Moreover, 34 chela