Regulation of DMT1 (non IRE isoform) by MicroRNA LET-7D

DMT1 (Nramp2/DCT1), also called SLC11A2, is a widely expressed metal-iron transporter that is vital for iron absorption (enterocytes) and utilization (erythroid precursors). Transcription of the mammalian SLC11A2 genes coding for DMT1 gives rise to four variant mRNA transcripts that differ in their tissue distribution and regulation. A 5′-end mRNA processing gives two isoforms: the first one starts from exon 1A, and the other from exon 1B. Whereas the 1B isoform is ubiquitous, the 1A isoform is tissue-specific, expressed predominantly in the duodenum and kidney. In addition, variable 3′-end processing yields two transcripts that differ in their 3′-translated regions and UTRs (untranslated regions). One of the transcripts, denoted IRE, contains in its 3′-UTR an iron-responsive element (IRE) that may alter mRNA stability according to iron status, so that the IRE-containing and non-IRE forms may be expected to differ mainly in their regulation by iron. In humans, DMT1 mutation causes microcytic hypochromic anemia due to decreased erythroid iron utilization with liver iron overload, high transferrin saturation and mildly elevated serum ferritin. MicroRNAs (miRNAs) are single-stranded RNAs of ~22 nucleotides in length, and they constitute a novel class of gene regulators. In animals, miRNAs exert their regulatory effects by binding to imperfect complementary sites within the 3′-untranslated regions (3′-UTRs) of their mRNA targets affecting protein translation. They are involved in a variety of important biological processes, such as developmental timing and patterning, apoptosis, hematopoietic differentiation, cell proliferation, organ development and tumorigenesis. The aim of our work was to study the microRNAs regulation of the gene involved in iron metabolism, particularly the microRNAs that target the DMT1 non IRE isoform gene. In-silico analysis of the mirBase targets database (Griffiths-Jones, 2004) was directed towards the identification of miRNAs potentially targeting DMT1 (non IRE isoform). Among others, we selected microRNA LET-7D because it was downregulated during eritroid differentiation of CD34+ cells and K562 cell line while DMT1 was upregulated, furthermore it belongs to a conserved microRNAs family. The over expression of the pre-microRNA LET-7D in K562 cell line down regulated both the mRNA and the protein expression of DMT1 (non IRE isoform). On the other hand, the DMT1 (IRE isoform) was up regulated. As control, we mutated the seed region of microRNA and the over expression of mutated microRNA LET-7D in K562 cell line did not interfere with DMT1 expression. In order to evaluate whether DMT1 was effectively a target of miR-LET-7D, the DMT1 3′UTR was cloned downstream of a luciferase reporter gene vector; the K562 cells line were then transfected with the over expressing vector and the reporter construct, with the relative luciferase activity showing that miRNA LET- 7D transfection led to decreased activity of the reporter gene, thus indicating binding with the 3′UTR and destabilisation of productive translation of luciferase mRNA. As controls, DMT1 3′UTR mutated in the miRNA LET-7D binding site was not affected by miRNA LET-7D. We are performing functional study to evaluate the state of iron metabolism in K562 cell line (stable clone) overexpressing the microRNA LET-7D. Here, we identify the microRNA LET-7D as a regulator of the iron metabolism through its targeting of the DMT1 (non IRE isoform).