Increase of microRNA-210, Decrease of Raptor Gene Expression and Alteration of Mammalian Target of Rapamycin Regulated Proteins following Mithramycin Treatment of Human Erythroid Cells

<div><p>Expression and regulation of microRNAs is an emerging issue in erythroid differentiation and globin gene expression in hemoglobin disorders. In the first part of this study microarray analysis was performed both in mithramycin-induced K562 cells and erythroid precursors from healthy subjects or β-thalassemia patients producing low or high levels of fetal hemoglobin. We demonstrated that: (a) microRNA-210 expression is higher in erythroid precursors from β-thalassemia patients with high production of fetal hemoglobin; (b) microRNA-210 increases as a consequence of mithramycin treatment of K562 cells and human erythroid progenitors both from healthy and β-thalassemia subjects; (c) this increase is associated with erythroid induction and elevated expression of γ-globin genes; (d) an anti-microRNA against microRNA-210 interferes with the mithramycin-induced changes of gene expression. In the second part of the study we have obtained convergent evidences suggesting raptor mRNA as a putative target of microRNA-210. Indeed, microRNA-210 binding sites of its 3’-UTR region were involved in expression and are targets of microRNA-210-mediated modulation in a luciferase reporter assays. Furthermore, (i) raptor mRNA and protein are down-regulated upon mithramycin-induction both in K562 cells and erythroid progenitors from healthy and β-thalassemia subjects. In addition, (ii) administration of anti-microRNA-210 to K562 cells decreased endogenous microRNA-210 and increased raptor mRNA and protein expression. Finally, (iii) treatment of K562 cells with premicroRNA-210 led to a decrease of raptor mRNA and protein. In conclusion, microRNA-210 and raptor are involved in mithramycin-mediated erythroid differentiation of K562 cells and participate to the fine-tuning and control of γ-globin gene expression in erythroid precursor cells.</p></div

Phenotype and genotype of the ErPCs from thalassemia patients employed in microRNA-210 and raptor analyses.

<p>(*) fold increase in MTH-treated Th-ErPCs (with respect to untreated control cells)</p><p>Phenotype and genotype of the ErPCs from thalassemia patients employed in microRNA-210 and raptor analyses.</p

Fold increase of the genes involved in the significant pathways obtained by gene expression profiling.

<p>The values referred to untreated K562 cells control.</p><p>¹. HBA1 (hemoglobin alpha 1), HBA2 (hemoglobin alpha 2), HBE1 (hemoglobin epsilon 1), HBZ (hemoglobin zeta), ERAF (erythroid associated factor), CPOX (coproporphyrinogen oxidase), HMBS (hydroxymethylbilane synthase), UROS (uroporphyrinogen III synthase), UROD (uroporphyrinogen decarboxylase), FECH (ferrochelatase)</p><p>². NFE2L3 nuclear factor erythroid-derived 2-like 3), EPB49 (erythrocyte membrane protein band 4.9), SLC4A1 (solute carrier family 4, anion exchanger, member 1 erythrocyte membrane protein band 3, Diego blood group).</p><p>Fold increase of the genes involved in the significant pathways obtained by gene expression profiling.</p

Expression of microRNA-210 and raptor mRNA in erythroid precursor cells.

<p>Quantification by RT-qPCR of microRNA-210 (A, C) and raptor mRNA (B, D) in 9 healthy donors (US) (A, B) and 9 β-thalassemia patients (Th) (C, D). The untreated ErPC samples are depicted as circles and the treated (30 nM MTH) as squares; samples from a same subject are identified by the same color. Means and standard deviations are reported. RT-qPCR analysis was performed after four days of erythroid differentiation. MicroRNA-let-7c (equally expressed in these samples) and 18S were used as internal reference genes to normalize RNA input.</p

Expression of transferrin receptor (TR) and Glycophorin A (GYPA) in ErPCs analyzed by flow cytometry.

<p>(A) FACS analysis of TR (green) and GYPA (red) antibody binding to ErPCs isolated after day 4 and day 8 of cell culture. (B) Relationship between TR and GYPA expression in day 4 ErPCs cultures. (C) Comparison of TR+/GYPA+ and B+ (benzidine-staining positive) cells in ErPCs from different β-thalassemic patients (n = 5, p<0.05).</p

MicroRNAs modulated in ErPCs from β-thalassemia patients and healthy donors and in K562 cells induced to erythroid differentiation by MTH.

<p>(A) Hierarchical clustering of ErPC samples from β-thalassemia patients and healthy donors (unaffected subjects, US). These samples were clustered according to the expression profile of the 43 microRNAs differentially expressed in samples from β-thalassemia patients with high HbF (Th17a, Th17b, ThFe37), with low HbF (ThFe30, ThFe42, ThFe52) and healthy donors (d1-US, d2-US). Different sample subtypes are indicated with color bars (red, high HbF; blue, US and low HbF). The differential expression of microRNAs can be easily followed by a color-code approach, indicating with green low levels, and with red high levels of microRNA content. (B) Heat map of the 71 microRNAs up- or down-regulated in K562 cells following MTH treatment. (-) = K562 cells untreated control. (C) Common microRNAs modulated in Th (high HbF) <i>vs</i>. Th (low HbF) plus US samples and in MTH-induced K562 <i>vs</i>. uninduced cells. (D) MicroRNA-210 expression analyzed by RT-qPCR in RNA samples from two Th (high HbF) ErPCs and three Th (low HbF) ErPCs. The data are represented as fold change with respect to ErPCs from healthy subjects (* = p<0.05; ** = p<0.01). (E) Changes of microR-210 (black) and γ-globin mRNA (white) expression in MTH-induced K562 cells in five independent experiments (* = p<0.05). The data represent fold change with respect to control untreated K562 cells. MicroRNA-let-7c (equally expressed in the samples) and 18S were used as internal reference gene sequences to normalize RNA input. (F) Correlation between microRNA-210 content in ®-thalassemia patient ErPCs (analyzed by RT-qPCR with respect to healthy subjects) and %HbF (analyzed by HPLC) in peripheral blood.</p

Effects of MTH treatment on ErPCs.

<p>(A) Modulation of microRNA-210, raptor, α- and γ-globin mRNAs in ErPCs from β-thalassemia patients by MTH. ErPCs obtained from 9 patients were grown in the two-phase liquid culture system. The effects were investigated in cells treated with 30 nM MTH for four days. The mRNA content was assayed by RT-qPCR using microRNA-let-7c (equally expressed in the samples) and 18S as reference gene sequences. The data represent mean±S.D. from 9 different experiments and are expressed as fold change with respect to untreated ErPCs (p<0.01, using One-way ANOVA, Kruskal-Wallis test). (B) Western blotting analysis of raptor protein in ErPCs from β-thalassemia patients, either untreated (-) or treated for four days with 30 nM MTH (p<0.05). The data represent the mean±S.D. of densitometric analysis from three independent experiments. In the upper insert, a representative example of Western blotting analysis.</p

Modulated genes following anti-microRNA-210 treatment analyzed by gene-profiling and involved in heme biosynthetic process and hemoglobin pathway.

<p>The samples analyzed were obtained respectively from untreated K562 cells (a) or K562 cells treated with MTH (30 nM) + anti-microRNA-210 (b), MTH (c), or MTH + anti-microRNA-588 (d) (p<0.05). The differential expression of microRNAs can be easily followed by a color-bar approach indicating low levels (green) and high levels (red) of mRNA content.</p

Effects of anti-microRNA-210 and premicroRNA-210 on K562 cells.

<p>(A) Effects of anti-microRNA-210 and premicroRNA-210 administration at the concentration of 200 nM with the siPort NeoFX transfection reagent (Ambion). The cells were seeded at 30000/ml and RNA extraction was carried out after five days of treatment. RT-qPCR analysis of microRNA-210, raptor and γ-globin transcripts was performed using untreated cells as reference and microRNA-let-7c and 18S as internal control gene sequences. The data represent the mean±S.D. from three independent experiments. (B) Effects of anti-microRNA-210 administration on raptor protein content in uninduced K562 cells and analyzed by Western blotting. The data represent the mean±S.D. from three independent experiments. (C) Effects of anti-microRNA-210 and premicroRNA-210 administration on raptor protein content in K562 cells analyzed by Western blotting. Beta-actin was used as the internal control. (D) Densitometric Western blotting analysis (as that shown in panel C) reporting the ratio raptor/β-actin. The data represent the mean±S.D. from three independent experiments (* = p<0.05; ** = p<0.01). (E) Analysis of microRNA-210, raptor and γ-globin transcripts in K562 cells induced to erythroid differentiation by MTH and treated with anti-microRNA-210 or with anti-microRNA-588 (taken as negative control). RT-qPCR data are presented as fold changes in respect to MTH-treated cells, using microRNA-let-7c and 18S as reference gene sequences. The data represent the mean±S.D. from three independent experiments.</p

Expression of microRNA-210 and raptor mRNA in K562 cells.

<p>Quantification by RT-qPCR of microRNA-210 (A) and raptor mRNA (B) in K562 cell line was performed. Samples from uninduced cells are presented as circles, from 30 nM MTH-treated cells as squares and from cells treated with MTH + anti-microRNA-210 as triangles. RT-qPCR analysis was performed after five days of erythroid differentiation. The three different experiments performed are indicated in black, grey and white (* = p<0.05). Mean and standard deviation are reported. MicroRNA-let-7c (equally expressed in the samples) and 18S were used as internal reference genes to normalize RNA input.</p