Fig 1.
Expression of transferrin receptor (TR) and Glycophorin A (GYPA) in ErPCs analyzed by flow cytometry.
(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).
Fig 2.
MicroRNAs modulated in ErPCs from β-thalassemia patients and healthy donors and in K562 cells induced to erythroid differentiation by MTH.
(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) vs. Th (low HbF) plus US samples and in MTH-induced K562 vs. 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.
Fig 3.
Modulation of gene expression by anti-microRNA-210 in K562 cells induced to erythroid differentiation by MTH.
(A) Heat map representation of 748 mRNAs clustered according to the expression profile. Cell cultures were carried out for six days. The samples analyzed were obtained from untreated K562 cells and K562 cells treated with MTH (30 nM) + anti-microRNA-210, MTH, or MTH + anti-microRNA-588 (taken as anti-microRNA-control). (B) RT-qPCRs of microRNA-210 in K562 cells treated with anti-microRNA-210 (white histogram), K562 cells induced with 30 nM MTH in the absence (black histogram) and in the presence of anti-microRNA-210 or anti-microRNA-588 (grey histograms). The cells were seeded at 10000 cells/500 μl and 30 nM MTH added at the first day of culture after anti-microRNA treatment. The data depicted represent relative fold increase with respect to untreated cells (mean±S.D. from four independent experiments). MicroRNA-let-7c was used as internal reference gene to normalize RNA input. (C) RT-qPCRs of γ-globin mRNA and (D) proportion of benzidine-positive cells (containing Hb) determined in untreated K562 cells (white histogram) and K562 cells treated with 30 nM MTH in the absence (black histogram) and in the presence of anti-microRNAs (grey histograms: anti-microRNA-210 or anti-microRNA-588, as indicated).
Table 1.
Fold increase of the genes involved in the significant pathways obtained by gene expression profiling.
Fig 4.
Modulated genes following anti-microRNA-210 treatment analyzed by gene-profiling and involved in heme biosynthetic process and hemoglobin pathway.
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.
Fig 5.
Raptor gene as a putative target of microRNA-210.
(A) Genes putatively modulated in gene expression analysis by microRNA-210 in human erythroid induced cells. Representation of the intersections of microRNA-210 targets whose expression is down-regulated in ErPCs from Th17 (high HbF) vs. Th1 (low HbF) and down-regulated in MTH-induced K562 cells, but resulting up regulated by anti-microRNA-210. Arrowed three putative microRNA-210 target genes. (B) Location of the two microRNA-210 two binding sites in the 3’-UTR of raptor mRNA. (C) Structure of microRNA-210 and the predicted alignments with the 3’-UTR sequence of raptor mRNA and with three validated target mRNAs (PLK1, ROD1, E2F3).
Fig 6.
Quantification of microRNA-210, raptor, α- and γ-globin transcripts in MTH-induced K562 cells.
(A) K562 cells were seeded at 30000/ml following treatment with 30 nM MTH and recovered at the indicated times. The raptor mRNA content is depicted as white symbols and γ-globin mRNA as black symbols. Untreated cells are depicted as squares and MTH-treated cells as circles. Results represent the fold changes with respect to untreated K562 cells at time = 0. (B) qPCR analysis of microRNA-210 (white histogram), raptor mRNA (black histogram), α- and γ-globin transcripts (grey histograms) in MTH-induced K562 cells after five days of erythroid differentiation (mean±S.D. from three different experiments). Results represent fold changes with respect untreated K562 cells. (C) K562 cells were treated with 30 nM MTH and 1 μM RAPA and analyzed after five days of induction. MicroRNA-210 and raptor mRNA content was calculated with respect to untreated cells using U6 snRNA or 18S as reference genes respectively (mean±S.D. from three different experiments). (D) Western blotting of the cellular extracts obtained from untreated K562 cells or K562 cells treated using 30 nM MTH and 1 μM RAPA for five days. Western Blotting was performed using antibodies against raptor protein (arrowed); in the lower part of panel D, the graph obtained by densitometric analysis of the autoradiograms is shown. Normalized intensity values are expressed as a percentage and mean (black) and standard deviation (white) from three independent experiments are reported. Ponceau S staining of the Western blots was used as the loading control.
Fig 7.
Expression of microRNA-210 and raptor mRNA in erythroid precursor cells.
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.
Table 2.
Phenotype and genotype of the ErPCs from thalassemia patients employed in microRNA-210 and raptor analyses.
Fig 8.
Expression of microRNA-210 and raptor mRNA in K562 cells.
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.
Fig 9.
The raptor microRNA-210 sites modulate luciferase activity in a reporter gene construct.
(A) Scheme of the pmirGLO vector, location and sequences of the cloned normal and mutated microRNA-210 sites in the raptor 3’-UTR. The arrowed nucleotides indicate the base substitution introduced in raptor target microRNA-210 mutated sites 1 and 2. (B) Luc2/hRluc ratios obtained in extracts of K562 cells treated with the pmirGLO vector or with the raptor-210-site1-pmiRGLO, MUT-raptor-210-site1-pmiRGLO, raptor-210-site2-pmiRGLO, MUT-raptor-210-site2-pmiRGLO vectors. (C) Relative luciferase activity in extracts treated with the raptor-210-site1-pmiRGLO, MUT-raptor-210-site1-pmiRGLO, raptor-210-site2-pmiRGLO, MUT-raptor-210-site2-pmiRGLO vectors in the presence of premicroRNA-210. The data represent the luciferase activity relative to K562 cells treated with MUT-raptor-210-site2-pmiRGLO vector.
Fig 10.
Comparison of the effects of MTH to RAPA on p70S6Kinase phosphorylation.
(A-D) K562 cells were treated as indicated with 30 nM MTH and 1 μM RAPA and analyzed after 2, 4, 24, 48 hours by Western blotting using monoclonal antibodies against p70S6Kinase (A, C) and against the phosphorylation site of p-p70S6K Thr389 (B, D); (-) = untreated cells. (E, F) Determination of raptor protein production using specific monoclonal antibodies in untreated K562 cells and in K562 cells treated with MTH for 24 and 48 hours (when the modification of p70S6Kinase phosphorylation occurs). Western blotting was performed using antibodies against raptor protein (arrowed). G, H) Western blotting showing expression of mTOR, used as loading control in addition to Ponceau S staining. (I, L) The densitometric analysis of K562 cells were treated as indicated with 30 nM MTH and 1 μM RAPA and analyzed after 2, 4, 24, 48 hours by Western blotting using monoclonal antibodies against the phosphorylation site of p-S6RP Ser235/236. The quantitative densitometric analysis of all the autoradiograms is shown in the lower part of the panels. Normalized intensity values are expressed as the mean (black) and standard deviation (white) from three independent experiments.
Fig 11.
Effects of MTH treatment on ErPCs.
(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.
Fig 12.
Effects of anti-microRNA-210 and premicroRNA-210 on K562 cells.
(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.
Fig 13.
Effects of siRNA against raptor mRNA on K562 cells γ-globin gene expression.
(A) Treatment of uninduced K562 cells with siRNA against raptor mRNA (black boxes) and siRNA against GAPDH mRNA (white boxes). (B) Effects of siRNA against raptor mRNA and siRNA against GAPDH mRNA on K562 cells induced to erythroid differentiation using a suboptimal concentration of MTH (15 nM). K562 cells were seeded at 10000 cell/100 μl following the Ambion transfection protocol using siPort NeoFX transfection reagent and 200 nM Silencer GAPDH siRNA and pre-designed siRNAs targeting raptor mRNA. Cells-to-cDNA II Kit was used to produce cDNA and RT-qPCR were conduced as reported in the methods section. The histograms represent the effects of si-GAPDH and si-raptor mRNA on GAPDH (white boxes), raptor (black boxes) and γ-globin (grey boxes) transcripts. The data represent the mean±S.D. from three independent experiments.