MicroRNA mediates post‐transcriptional expression of human lactoferrin receptor (hLfR)

MicroRNAs (miRNAs) are small, non‐coding RNA molecules typically 19–25 nucleotides (nt) in size, and their function as gene expression modulators is critical for mammalian development. Lactoferrin receptor (LfR) on the enterocyte apical membrane has been suggested to play a key role in the absorption of lactoferrin‐bound iron from breast milk during infancy. The objective of this study was to identify the miRNA regulating hLfR mRNA translation. Sequence analysis revealed the presence of a conserved region complementary to the seed region of hsa‐miR‐584 (nt 2–8). The 120 bp hLfR‐3′UTR was cloned into pGL3‐control luciferase reporter vector. Site‐directed mutagenesis was introduced to 3'UTR sequence corresponding to miRNA seed region. The vectors were co‐transfected with hsa‐miR‐584 mimic into HEK293 cells. Furthermore, hsa‐miR‐584 mimic/inhibitor was transfected into Caco‐2 cells to evaluate its capacity to regulate endogenous hLfR expression examined by western blot and Q‐PCR. By luciferase reporter assay, we determined that hsa‐miR‐584 repressed the reporter dose‐dependently, and repression was specific as hsa‐miR‐584 did not repress the reporter once the seed region was mutated, and neither could a structurally unrelated miRNA (cel‐miR‐67). The repression was hLfR‐3'UTR dependent, as hsa‐miR‐584 did not affect pGL3‐control reporter. We also determined that hsa‐miR‐584 decreased endogenous hLfR protein expression, without altering the mRNA level. Taken together, our findings demonstrate that hsa‐miR‐584 mediates the post‐transcriptional expression of hLfR by inhibiting mRNA activity. These findings suggest a novel mechanism by which nutrient absorption is regulated by microRNA.