Hydroxyurea down‐regulates BCL11A, KLF‐1 and MYB through miRNA‐mediated actions to induce γ‐globin expression: implications for new therapeutic approaches of sickle cell disease
Abstract:Background
The major therapeutic benefit of hydroxyurea, the only FDA-approved pharmacologic treatment for sickle cell disease (SCD), is directly related to fetal hemoglobin (HbF) production that leads to significant reduction of morbidity and mortality. However, potential adverse effects such as infertility, susceptibility to infections, or teratogenic effect have been subject of concerns. Therefore, understanding HU molecular mechanisms of action, could lead to alternative therapeutic agents to … Show more
“…Our miRNAs data analysis showed abnormal expression of a pool of upregulated erythroid‐miRNAs that have not yet been discussed in the anti‐doping field. This pool is involved in erythroid differentiation and hematopoiesis, induction of HbF and the transcriptional regulation of the human γ‐globin gene, and previous studies confirmed the significant involvement of a network between miRNAs and transcription factors in HbF production by erythroid cells …”
BACKGROUND: Autologous blood transfusion (ABT) is a performance-enhancing method prohibited in sport; its detection is a key issue in the field of anti-doping. Among novel markers enabling ABT detection, microRNAs (miRNAs) might be considered a promising analytical tool.
STUDY DESIGN AND METHODS:We studied the changes of erythroid-related microRNAs following ABT, to identify novel biomarkers. Fifteen healthy trained males were studied from a population of 24 subjects, enrolled and randomized into a Transfusion (T) and a Control (C) group. Seriated blood samples were obtained in the T group before and after the two ABT procedures (withdrawal, with blood refrigerated or cryopreserved, and reinfusion), and in the C group at the same time points. Traditional hematological parameters were assessed. Samples were tested by microarray analysis of a pre-identified set of erythroid-related miRNAs.
RESULTS:Hematological parameters showed moderate changes only in the T group, particularly following blood withdrawal. Among erythroid-related miRNAs tested, following ABT a pool of 7 miRNAs associated with fetal hemoglobin and regulating transcriptional repressors of gamma-globin gene was found stable in C and differently expressed in three out of six T subjects in the completed phase of ABT, independently from blood conservation. Particularly, two or more erythropoiesis-related miRNAs within the shortlist constituted of miR- 126-3p, miR-144-3p, miR-191-3p, miR-197-3p, miR-486-3p, miR-486-5p, and miR-92a-3p were significantly upregulated in T subjects after reinfusion, with a person-to-person variability but with congruent changes.
CONCLUSIONS:This study describes a signature of potential interest for ABT detection in sports, based on the analysis of miRNAs associated with erythroid features.A utologous blood transfusion (ABT) is a performance-enhancing method prohibited in sport, 1 with its detection a key issue in the anti-doping field. Given the lack of a method for its direct detection, 2-5 an approach to the indirect detection of ABT has been considered. The athlete biological passport (ABP) hematological module 6-8 has been adopted by the World Anti-Doping Agency (WADA) to identify and, when necessary, sanction athletes showing abnormal changes in hematological parameters. Novel markers enabling ABT indirect detection have also been proposed, [2][3][4][9][10][11][12][13] including those derived from genomics applied to microRNAs (miRNAs), and are considered a promising analytical tool. [14][15][16][17] In this respect, miRNAs are short non-coding RNA molecules, which act as gene regulators by repressing translation or by inducing the cleavage of target RNA transcripts. [18][19][20][21] Recent evidence suggests that miRNAs (including miRNAs present as extracellular molecules in plasma) can be considered a From the
“…Our miRNAs data analysis showed abnormal expression of a pool of upregulated erythroid‐miRNAs that have not yet been discussed in the anti‐doping field. This pool is involved in erythroid differentiation and hematopoiesis, induction of HbF and the transcriptional regulation of the human γ‐globin gene, and previous studies confirmed the significant involvement of a network between miRNAs and transcription factors in HbF production by erythroid cells …”
BACKGROUND: Autologous blood transfusion (ABT) is a performance-enhancing method prohibited in sport; its detection is a key issue in the field of anti-doping. Among novel markers enabling ABT detection, microRNAs (miRNAs) might be considered a promising analytical tool.
STUDY DESIGN AND METHODS:We studied the changes of erythroid-related microRNAs following ABT, to identify novel biomarkers. Fifteen healthy trained males were studied from a population of 24 subjects, enrolled and randomized into a Transfusion (T) and a Control (C) group. Seriated blood samples were obtained in the T group before and after the two ABT procedures (withdrawal, with blood refrigerated or cryopreserved, and reinfusion), and in the C group at the same time points. Traditional hematological parameters were assessed. Samples were tested by microarray analysis of a pre-identified set of erythroid-related miRNAs.
RESULTS:Hematological parameters showed moderate changes only in the T group, particularly following blood withdrawal. Among erythroid-related miRNAs tested, following ABT a pool of 7 miRNAs associated with fetal hemoglobin and regulating transcriptional repressors of gamma-globin gene was found stable in C and differently expressed in three out of six T subjects in the completed phase of ABT, independently from blood conservation. Particularly, two or more erythropoiesis-related miRNAs within the shortlist constituted of miR- 126-3p, miR-144-3p, miR-191-3p, miR-197-3p, miR-486-3p, miR-486-5p, and miR-92a-3p were significantly upregulated in T subjects after reinfusion, with a person-to-person variability but with congruent changes.
CONCLUSIONS:This study describes a signature of potential interest for ABT detection in sports, based on the analysis of miRNAs associated with erythroid features.A utologous blood transfusion (ABT) is a performance-enhancing method prohibited in sport, 1 with its detection a key issue in the anti-doping field. Given the lack of a method for its direct detection, 2-5 an approach to the indirect detection of ABT has been considered. The athlete biological passport (ABP) hematological module 6-8 has been adopted by the World Anti-Doping Agency (WADA) to identify and, when necessary, sanction athletes showing abnormal changes in hematological parameters. Novel markers enabling ABT indirect detection have also been proposed, [2][3][4][9][10][11][12][13] including those derived from genomics applied to microRNAs (miRNAs), and are considered a promising analytical tool. [14][15][16][17] In this respect, miRNAs are short non-coding RNA molecules, which act as gene regulators by repressing translation or by inducing the cleavage of target RNA transcripts. [18][19][20][21] Recent evidence suggests that miRNAs (including miRNAs present as extracellular molecules in plasma) can be considered a From the
“…Human leukemia K562 [ 38 , 39 ] and K562(BCL11A-XL) cells [ 41 , 49 , 50 ] were cultured in humified atmosphere of 5% CO 2 /air in RPMI 1640 medium (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (Biowest, Nuaillé, France), 50 U/mL penicillin and 50 μg/mL streptomycin. Mithramycin (MTH) [ 51 , 52 ], rapamycin (RAPA) [ 53 ], resveratrol (RVS) [ 54 ] and hydroxyurea (HU) [ 55 ] were purchased from Sigma. Treatment of cells with erythroid differentiation inducers was carried out by adding the appropriate drug concentrations at the beginning of the cultures (cells were seeded at 30,000/mL).…”
The involvement of microRNAs in the control of repressors of human γ-globin gene transcription has been firmly demonstrated, as described for the miR-486-3p mediated down-regulation of BCL11A. On the other hand, we have reported that miR-210 is involved in erythroid differentiation and, possibly, in γ-globin gene up-regulation. In the present study, we have identified the coding sequence of BCL11A as a possible target of miR-210. The following results sustain this hypothesis: (a) interactions between miR-210 and the miR-210 BCL11A site were demonstrated by SPR-based biomolecular interaction analysis (BIA); (b) the miR-210 site of BCL11A is conserved through molecular evolution; (c) forced expression of miR-210 leads to decrease of BCL11A-XL and increase of γ-globin mRNA content in erythroid cells, including erythroid precursors isolated from β-thalassemia patients. Our study suggests that the coding mRNA sequence of BCL11A can be targeted by miR-210. In addition to the theoretical point of view, these data are of interest from the applied point of view, supporting a novel strategy to inhibit BCL11A by mimicking miR-210 functions, accordingly with the concept supported by several papers and patent applications that inhibition of BCL11A is an efficient strategy for fetal hemoglobin induction in the treatment of β-thalassemia.
“…6 ), in consideration of the fact that BCL11A is a transcriptional repressor of γ-globin genes. This is of interest, since inhibition of BCL11A is a recognized strategy for HbF induction for treatment of β-thalassemia, as suggested by several papers and patent applications [ 110 , 173 ]. Fig.…”
Section: Examples Of Non-coding Rnas In Rare Genetic Diseasesmentioning
Since the discovery and classification of non-coding RNAs, their roles have gained great attention. In this respect, microRNAs and long non-coding RNAs have been firmly demonstrated to be linked to regulation of gene expression and onset of human diseases, including rare genetic diseases; therefore they are suitable targets for therapeutic intervention. This issue, in the context of rare genetic diseases, is being considered by an increasing number of research groups and is of key interest to the health community. In the case of rare genetic diseases, the possibility of developing personalized therapy in precision medicine has attracted the attention of researchers and clinicians involved in developing “orphan medicinal products” and proposing these to the European Medicines Agency (EMA) and to the Food and Drug Administration (FDA) Office of Orphan Products Development (OOPD) in the United States. The major focuses of these activities are the evaluation and development of products (drugs, biologics, devices, or medical foods) considered to be promising for diagnosis and/or treatment of rare diseases or conditions, including rare genetic diseases. In an increasing number of rare genetic diseases, analysis of microRNAs and long non-coding RNAs has been proven a promising strategy. These diseases include, but are not limited to, Duchenne muscular dystrophy, cystic fibrosis, Rett syndrome, and β-thalassemia. In conclusion, a large number of approaches based on targeting microRNAs and long non-coding RNAs are expected in the field of molecular diagnosis and therapy, with a facilitated technological transfer in the case of rare genetic diseases, in virtue of the existing regulation concerning these diseases.
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