Regulation of divalent metal transporter 1 (DMT1) non-IRE isoform by the microRNA Let-7d in erythroid cells.
BACKGROUND
Divalent metal transporter 1 (DMT1) is a widely expressed metal-iron transporter gene encoding four variant mRNA transcripts, differing for alternative promoter at 5' (DMT1 1A and 1B) and alternative splicing at 3' UTR, differing by a specific sequence either containing or lacking an iron regulatory element (+IRE and -IRE, respectively). DMT1-IRE might be the major DMT1 isoform expressed in erythroid cells, although its regulation pathways are still unknown.


METHODS
The microRNA (miRNA) Let-7d (miR-Let-7d) was selected by the analysis of four miRNAs, predicted to target the DMT1-IRE gene in CD34(+) hematopoietic progenitor cells, in K562 and in HEL cells induced to erythroid differentiation. Using a luciferase reporter assay we demonstrated the inhibition of DMT1-IRE by miR-Let-7d in K562 and HEL cells. The function of miR-Let-7d in erythroid cells was evaluated by the flow cytometry analysis of erythroid differentiation markers, by benzidine staining and by iron flame atomic absorption for the evaluation of iron concentration in the endosomes from K562 cells over-expressing miR-Let-7d.


RESULTS
We show that in erythroid cells, DMT1-IRE expression is under the regulation of miR-Let-7d. DMT1-IRE and miR-Let-7d are inversely correlated with CD34(+) cells, K562 and HEL cells during erythroid differentiation. Moreover, overexpression of miR-Let-7d decreases the expression of DMT1-IRE at the mRNA and protein levels in K562 and HEL cells. MiR-Let-7d impairs erythroid differentiation of K562 cells by accumulation of iron in the endosomes.


CONCLUSIONS
Overall, these data suggest that miR-Let-7d participates in the finely tuned regulation of iron metabolism by targeting DMT1-IRE isoform in erythroid cells.
Andolfo
Immacolata
I
CEINGE, Biotecnologie Avanzate, Via, Comunale Margherita 482, 80145 Naples, Italy.
De Falco
Luigia
L
Asci
Roberta
R
Russo
Roberta
R
Colucci
Simona
S
Gorrese
Marisa
M
Zollo
Massimo
M
Iolascon
Achille
A
D020413:3' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D027682:Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D011992:Endosomes / Q000378:metabolism; D041905:Erythroid Cells / Q000378:metabolism*; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007501:Iron / Q000378:metabolism; D020014:K562 Cells; D035683:MicroRNAs / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D000099236:Solute Carrier Family 11, Member 2
D020413:3' Untranslated Regions; D027682:Cation Transport Proteins; D007501:Iron; D035683:MicroRNAs; D020033:Protein Isoforms; D012333:RNA, Messenger; D000099236:Solute Carrier Family 11, Member 2; C487931:mirnlet7 microRNA, human
FEBS Lett. 2005 Mar 28;579(9):1923-9
15792797
Nat Genet. 2007 Oct;39(10):1278-84
17893677
Blood. 2008 Nov 15;112(10):4268-75
18776082
Cell. 2007 Jun 15;129(6):1141-51
17524464
Blood. 2004 May 15;103(10):3991-2
15121718
Am J Physiol Gastrointest Liver Physiol. 2005 Dec;289(6):G981-6
16286504
Nature. 1997 Jul 31;388(6641):482-8
9242408
Blood. 2006 May 15;107(10):4168-70
16439678
Blood. 2007 Jul 1;110(1):125-32
17376890
Genomics. 1995 Jan 20;25(2):514-25
7789986
Pathology. 2009;41(3):204-13
19291531
J Clin Invest. 2005 May;115(5):1258-66
15849611
Trends Cell Biol. 2008 Oct;18(10):505-16
18774294
Mol Cells. 2005 Feb 28;19(1):1-15
15750334
Proc Natl Acad Sci U S A. 2009 Sep 8;106(36):15489-94
19706893
Nat Rev Mol Cell Biol. 2008 Jan;9(1):72-81
17987043
Blood. 2006 Jan 1;107(1):349-54
16160008
Nat Genet. 1997 Aug;16(4):383-6
9241278
Nucleic Acids Res. 2004 Jan 1;32(Database issue):D109-11
14681370
Biochem J. 2006 Sep 15;398(3):539-46
16737442
Blood Cells Mol Dis. 1998 Jun;24(2):199-215
9642100
Mol Biol Cell. 2002 Dec;13(12):4371-87
12475959
Blood. 2001 Dec 15;98(13):3823-30
11739192
Proc Natl Acad Sci U S A. 1998 Feb 3;95(3):1148-53
9448300
Biochem Pharmacol. 2005 Jun 1;69(11):1647-55
15896344
Blood. 2005 Feb 1;105(3):1337-42
15459009
Biochem J. 2007 Apr 1;403(1):59-69
17109629
Blood. 2008 Jul 15;112(2):219-30
18606887
Nat Rev Genet. 2004 Jul;5(7):522-31
15211354
Proc Natl Acad Sci U S A. 2002 Sep 17;99(19):12345-50
12209011
Haematologica. 2009 Mar;94(3):395-408
19181781
Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18578-83
19011085
Science. 2003 Jul 18;301(5631):336-8
12869753
Cell Metab. 2009 Feb;9(2):152-64
19147412
Blood. 1999 Jun 15;93(12):4406-17
10361139
Nature. 2004 Sep 16;431(7006):350-5
15372042
Cell Metab. 2008 Jan;7(1):79-85
18177727
Nature. 2007 Jun 14;447(7146):823-8
17507929
Cell. 2004 Jan 23;116(2):281-97
14744438
Nat Methods. 2006 Nov;3(11):881-6
17060911