CEINGE, Biotecnologie Avanzate, Via, Comunale Margherita 482, 80145 Naples, Italy.
Haematologica. 2010 Aug;95(8):1244-52. doi: 10.3324/haematol.2009.020685. Epub 2010 Apr 21.
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.
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.
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.
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.
二价金属转运蛋白 1（DMT1）是一种广泛表达的金属铁转运蛋白基因，编码四种变体 mRNA 转录本，它们在 5'端的启动子（DMT1 1A 和 1B）和 3'UTR 的剪接方式不同，在 3'UTR 上通过特定序列的存在或缺失分别含有或缺乏铁调节元件（+IRE 和 -IRE）。DMT1-IRE 可能是红系细胞中表达的主要 DMT1 同工型，尽管其调节途径尚不清楚。
通过分析在 CD34+造血祖细胞、K562 细胞和诱导红细胞分化的 HEL 细胞中预测靶向 DMT1-IRE 基因的四种 miRNA，选择了 microRNA (miRNA) Let-7d (miR-Let-7d)。使用荧光素酶报告基因检测，我们证明了 miR-Let-7d 在 K562 和 HEL 细胞中对 DMT1-IRE 的抑制作用。通过流式细胞术分析红细胞分化标志物、联苯胺染色和铁火焰原子吸收法评估过表达 miR-Let-7d 的 K562 细胞内体中铁浓度，评估 miR-Let-7d 在红细胞中的功能。
我们表明，在红细胞中，DMT1-IRE 的表达受 miR-Let-7d 的调控。DMT1-IRE 和 miR-Let-7d 与 CD34+细胞、K562 细胞和 HEL 细胞在红细胞分化过程中呈负相关。此外，miR-Let-7d 的过表达降低了 K562 和 HEL 细胞中 DMT1-IRE 的 mRNA 和蛋白水平表达。miR-Let-7d 通过铁在内体中的积累，损害 K562 细胞的红细胞分化。
总之，这些数据表明，miR-Let-7d 通过靶向红细胞中的 DMT1-IRE 同工型参与铁代谢的精细调节。
