Abstract
Divalent metal transporter 1 (DMT1) is a key transporter of iron uptake and delivering in human and animals. However, post-transcriptional regulation of DMT1 is poorly understood. In this study, bioinformatic algorithms (TargetScan, PITA, miRanda, and miRDB) were applied to predict, screen, analyze, and obtain microRNA-16 family members (miR-16, miR-195, miR-497, and miR-15b) targeting DMT1, seed sequence and their binding sites within DMT1 3′ untranslated region (3′ UTR) region. As demonstrated by dual-luciferase reporter assays, luciferase activity of DMT1 3′ UTR reporter was impaired/enhanced when microRNA-16 family member over-expression plasmid/its inhibitor was transfected to HCT116 cells. Corroboratively, co-transfection of microRNA-16 family member over-expression plasmid and DMT1 3′ UTR mutant reporter repressed the luciferase activity in HCT116 cells. In addition, over-expression microRNA-16 family member augmented its expression and diminished DMT1 protein expression in HCT116 cells. Interestingly, tail vein injection of miR-16 assay revealed reduced plasma iron levels, higher miR-16 expression, and lower DMT1 protein expression in the duodenum of mice. Taken together, we provide evidence that microRNA-16 family (miR-16, miR-195, miR-497, and miR-15b) is confirmed to repress intestinal DMT1 expression in vitro and in vivo, which will give valuable insight into post-transcriptional regulation of DMT1.
Highlights
The divalent metal transporter 1 (DMT1), commonly abbreviated as Divalent metal transporter 1 (DMT1), is generally considered to be key iron transporter for intestinal ferrous (Fe2+) iron uptake, and delivering iron to peripheral tissues via transferrin (Fleming et al, 1998; Gunshin et al, 2005)
Bioinformatic analyses indicated that these microRNAs sequence and its binding sites within the DMT1 3 untranslated region (3 UTR) sequence are highly conserved among human, mouse, rat, and pig (Figure 1B)
Co-transfection experiments implied that microRNA-16 family member significantly enhanced luciferase reporter activities in the HCT116 cells treated with microRNA overexpression plasmid and DMT1 mutant reporter containing all the three mutated binding site (P < 0.01) (Figures 3B–E)
Summary
The divalent metal transporter 1 (DMT1), commonly abbreviated as DMT1, is generally considered to be key iron transporter for intestinal ferrous (Fe2+) iron uptake, and delivering iron to peripheral tissues via transferrin (Fleming et al, 1998; Gunshin et al, 2005). DMT1 plays a crucial role in iron absorption in the duodenum, where it is involved in dietary non-transferrin-bound iron uptake from the intestinal lumen (Fleming et al, 1997; Canonne-Hergaux et al, 1999). It is proposed that DMT1 mutant rodents exhibit microcytic, hypochromic anemia (Fleming et al, 1998; Canonne-Hergaux et al, 2000), whereas high-level expression of DMT1 contributes to neurodegenerative diseases (Salazar et al, 2008; Tian et al, 2018). A tight regulation of DMT1 expression is indispensable for maintenance of life in eukaryotes
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