The role of microRNA in the regulation of post-transcriptional expression of lactoferrin receptor (LfR) and its implication for regional development of the small intestine

In this study, using the piglet as a model, we first identified a small intestinal lactoferrin receptor (LfR) cDNA, and determined its mRNA and protein expression during different stages of intestinal development. The coding region of the pig LfR cDNA was cloned by PCR using conserved sequences among species. We determined the pig SI-LfR open reading frame (ORF), which consists of 972 bp, resulting in a protein with a molecular mass ∼135 kD and ∼35 kD under non-reducing and reducing conditions, respectively. Using real-time Q-PCR, we determined that LfR expression significantly increases with age in the duodenum and reciprocally decreases in the jejunum. Intestinal LfR protein expression was maintained at all time points in the jejunum; however, in the duodenum LfR abundance reached maximum levels at 6 months. In BBMV fractions, LfR abundance significantly increased with age. Taken together, our findings demonstrate the presence of a human SI-LfR homologue in the pig, with mRNA and protein expression concomitantly regulated in the duodenum and inversely regulated in the jejunum. These findings suggest a mechanism by which pig Lf can be internalized in the intestine, and that this receptor is under post-transcriptional regulation.;We therefore performed sequence analyses to examine putative post-transcriptional regulation mechanisms for LfR, and identified the presence of a conserved region in LfR mRNA-3'UTR complementary to the seed region of miR-584 (5' nt 2-8). miR-584 was further found to co-localize with LfR mRNA in the small intestine. The LfR mRNA-3'UTR was cloned into pGL3-control luciferase reporter vector. By luciferase reporter assay, miR-584 specifically repressed the reporter activity in a dose-dependent manner. miR-584 reduced endogenous LfR protein expression, without significantly affecting the mRNA level. We also determined by real-time Q-PCR that miR-584 is expressed in fetal mouse small intestine, most abundantly at gestational day 18.5, and decreases until postnatal day 7, whereas LfR mRNA was minimally expressed gestational day 18.5, and increases until postnatal day 7. Taken together, we propose that miR-584 mediates post-transcriptional expression of LfR during the perinatal period. These findings demonstrate a novel example of how microRNAs may be involved in regulation of nutrient metabolism in the newborn.;Having established the fact that the intestinal receptor for Lf absorption is regulated by specific miRNA, we further examined whether miRNAs are also involved in the downstream functions of Lf. As Lf has been demonstrated to stimulate intestinal cell proliferation and differentiation, we examined the role of miRNA in this process. The Wnt/beta-catenin/TCF4 pathway plays critical roles in the differentiation of small intestinal progenitor cells in the crypts to stably supply specialized villous cell lineages; however, downstream targets of beta-catenin/TCF4 complex have not been extensively characterized. We identified miR-30e as an immediate downstream target activated by beta-catenin/TCF4 complex during intestinal cell differentiation. miR-30e was detected in the peri-nuclear region of the crypt cell line IEC-6. Bioinformatics analysis revealed clustered beta-catenin/TCF4 binding sites within the miR-30e promoter region for human, mouse and rat. This promoter region was cloned into pGL3-control luciferase reporter vector, with the enhancer region removed. Transfection of pCMV-SPORT6-beta-catenin expression vector dose-dependently increased luciferase activity, and co-transfection of pCMV-SPORT6-TCF4 expression vector further enhanced the promoter activity. Dexamethasone induced differentiation of IEC-6 cells caused a 2.5-fold increase in miR-30e expression, and upon beta-catenin siRNA, miR-30e increased 1.3-fold. Electrophoretic mobility shift assay confirmed the binding between beta-catenin/TCF4 complexes from IEC-6 nuclear extracts and the putative sequences in the miR-30e promoter. These results demonstrate that beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation.;In conclusion, miRNAs define a new level of regulation for Lf biology, miR-584 regulates LfR expression for Lf absorption during a perinatal period, and beta-catenin/TCF4 transactivates miR-30e in Wnt singnalling pathway during intestinal cell differentiation. Evidence of involvement of miRNA in Lf biology provides valuable information to both understand mechanisms by which other milk components or nutrients exert their functions, and lead to an era when miRNAs serve as targets to correct illnesses related to nutrient metabolism. (Abstract shortened by UMI.)...