RESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causative of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 Spike protein (S-protein) plays an important role in the early phase of SARS-CoV-2 infection through efficient interaction with ACE2. The S-protein is produced by RNA-based COVID-19 vaccines, that were fundamental for the reduction of the viral spread within the population and the clinical severity of COVID-19. However, the S-protein has been hypothesized to be responsible for damaging cells of several tissues and for some important side effects of RNA-based COVID-19 vaccines. Considering the impact of COVID-19 and SARS-CoV-2 infection on the hematopoietic system, the aim of this study was to verify the effect of the BNT162b2 vaccine on erythroid differentiation of the human K562 cell line, that has been in the past intensively studied as a model system mimicking some steps of erythropoiesis. In this context, we focused on hemoglobin production and induced expression of embryo-fetal globin genes, that are among the most important features of K562 erythroid differentiation. We found that the BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation of K562 cells. Reverse-transcription-qPCR and Western blotting assays demonstrated that suppression of erythroid differentiation was associated with sharp inhibition of the expression of α-globin and γ-globin mRNA accumulation. Inhibition of accumulation of ζ-globin and ε-globin mRNAs was also observed. In addition, we provide in silico studies suggesting a direct interaction between SARS-CoV-2 Spike protein and Hb Portland, that is the major hemoglobin produced by K562 cells. This study thus provides information suggesting the need of great attention on possible alteration of hematopoietic parameters following SARS-CoV-2 infection and/or COVID-19 vaccination.
Assuntos
COVID-19 , Leucemia Eritroblástica Aguda , Humanos , Células K562 , Plicamicina/farmacologia , Plicamicina/metabolismo , Vacinas contra COVID-19/metabolismo , Vacina BNT162 , Leucemia Eritroblástica Aguda/metabolismo , COVID-19/prevenção & controle , COVID-19/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Hemoglobinas/metabolismo , RNA Mensageiro/genética , Células Eritroides/metabolismoRESUMO
In this short review, we presented and discussed studies on the expression of globin genes in ß-thalassemia, focusing on the impact of α-globin gene expression and α-globin modifiers on the phenotype and clinical severity of ß-thalassemia. We first discussed the impact of the excess of free α-globin on the phenotype of ß-thalassemia. We then reviewed studies focusing on the expression of α-globin-stabilizing protein (AHSP), as a potential strategy of counteracting the effects of the excess of free α-globin on erythroid cells. Alternative processes controlling α-globin excess were also considered, including the activation of autophagy by ß-thalassemia erythroid cells. Altogether, the studies reviewed herein are expected to have a potential impact on the management of patients with ß-thalassemia and other hemoglobinopathies for which reduction in α-globin excess is clinically beneficial.
Assuntos
Hemoglobinopatias , Talassemia beta , Humanos , Talassemia beta/genética , alfa-Globinas/genética , alfa-Globinas/metabolismo , Hemoglobinopatias/genética , Fenótipo , Expressão Gênica , Proteínas Sanguíneas/genética , Chaperonas Moleculares/genéticaRESUMO
Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. ß-Thalassemia). The objective of the present study was to analyse the activity of CD4+ and CD8+ T cells in ß-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4+ and CD8+ T cells, respectively. The main results of the present study are that treatment of ß-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4+ and CD8+ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that ß-Thalassemia patients are considered prone to immune deficiency.
Assuntos
Sirolimo , Talassemia beta , Idoso , Humanos , Talassemia beta/tratamento farmacológico , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Leucócitos Mononucleares , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Serina-Treonina Quinases TORRESUMO
The human homologue of mouse Ly-1 antibody reactive clone protein (LYAR) is a putative novel regulator of γ-globin gene transcription. The LYAR DNA-binding motif (5'-GGTTAT-3') is located within the 5'-UTR of the Aγ-globin gene. The LYAR rs368698783 (G>A) polymorphism is present in ß-thalassemia patients and decreases the LYAR binding efficiency to the Aγ-globin gene. The objective of this study was to stratify ß-thalassemia patients with respect to the rs368698783 (G>A) polymorphism and to verify whether their erythroid precursor cells (ErPCs) differentially respond in vitro to selected fetal hemoglobin (HbF) inducers. The rs368698783 (G>A) polymorphism was detected by DNA sequencing, hemoglobin production by HPLC, and accumulation of globin mRNAs by RT-qPCR. We found that the LYAR rs368698783 (G>A) polymorphism is associated with high basal and induced production of fetal hemoglobin in ß-thalassemia patients. The most striking association was found using rapamycin as an HbF inducer. The results presented here could be considered important not only for basic biomedicine but also in applied translational research for precision medicine in personalized therapy of ß-thalassemia. Accordingly, our data suggest that the rs368698783 polymorphism might be considered among the parameters useful to recruit patients with the highest probability of responding to in vivo hydroxyurea (HU) treatment.
Assuntos
Células Precursoras Eritroides , Talassemia beta , Humanos , Talassemia beta/tratamento farmacológico , Talassemia beta/genética , Talassemia beta/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Precursoras Eritroides/metabolismo , Hemoglobina Fetal/análise , gama-Globinas/genética , gama-Globinas/metabolismo , Proteínas Nucleares/genética , Polimorfismo GenéticoRESUMO
The ß-thalassemias are hereditary monogenic diseases characterized by a low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible for the ß-thalassemia-associated ineffective erythropoiesis. Therefore, the decrease in excess α-globin is a relevant clinical effect for these patients and can be realized through the induction of fetal hemoglobin, autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogs on the induction of fetal hemoglobin (HbF) are of key importance for therapeutic protocols in a variety of hemoglobinopathies, including ß-thalassemias. In this research communication, we report data showing that a decrease in autophagy-associated p62 protein, increased expression of ULK-1, and reduction in excess α-globin are occurring in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and a decrease in α-globin content were found in ErPCs isolated from ß-thalassemia patients recruited for the NCT03877809 clinical trial and treated with 0.5-2 mg/day sirolimus. Our data support the concept that autophagy, ULK1 expression, and α-globin chain reduction should be considered important endpoints in sirolimus-based clinical trials for ß-thalassemias.
Assuntos
Talassemia beta , Adulto , Humanos , Talassemia beta/tratamento farmacológico , Talassemia beta/genética , Talassemia beta/metabolismo , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Hemoglobina Fetal , alfa-Globinas/genética , alfa-Globinas/metabolismo , RNA Mensageiro/genética , Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Peptídeos e Proteínas de Sinalização Intracelular/genéticaRESUMO
Induction of fetal hemoglobin (HbF) is highly beneficial for patients carrying ß-thalassemia, and novel HbF inducers are highly needed. Here, we describe a new class of promising HbF inducers characterized by an isoxazole chemical skeleton and obtained through modification of two natural molecules, geldanamycin and radicicol. After preliminary biological assays based on benzidine staining and RT-qPCR conducted on human erythroleukemic K562 cells, we employed erythroid precursors cells (ErPCs) isolated from ß-thalassemic patients. ErPCs weretreated with appropriate concentrations of isoxazole derivatives. The accumulation of globin mRNAs was studied by RT-qPCR, and hemoglobin production by HPLC. We demonstrated the high efficacy of isozaxoles in inducing HbF. Most of these derivatives displayed an activity similar to that observed using known HbF inducers, such as hydroxyurea (HU) or rapamycin; some of the analyzed compounds were able to induce HbF with more efficiency than HU. All the compounds were active in reducing the excess of free α-globin in treated ErPCs. All the compounds displayed a lack of genotoxicity. These novel isoxazoles deserve further pre-clinical study aimed at verifying whether they are suitable for the development of therapeutic protocols for ß-thalassemia.
Assuntos
Hemoglobina Fetal , Talassemia beta , Humanos , Hemoglobina Fetal/genética , Células Precursoras Eritroides , Talassemia beta/tratamento farmacológico , Bioensaio , Hidroxiureia/farmacologia , IsoxazóisRESUMO
(1) Background: In the development of new and more effective anticancer approaches, combined treatments appear of great interest. Combination therapy could be of importance in the management of glioblastoma (GBM), a lethal malignancy that accounts for 42% of cancer of the central nervous system, with a median survival of 15 months. This study aimed to verify the activity on a glioblastoma cancer cell line of one of the most active compounds of a novel series of tubulin polymerization inhibitors based on the 1-(3',4',5'-trimethoxyphenyl)-2-aryl-1H-imidazole scaffold, used in combination with a miRNA inhibitor molecule targeting the oncomiRNA miR-10b-5p. This microRNA was selected in consideration of the role of miR-10b-5p on the onset and progression of glioblastoma. (2) Methods: Apoptosis was analyzed by Annexin-V and Caspase 3/7 assays, efficacy of the anti-miR-10b-5p was assessed by determining the miR-10b-5p content by RT-qPCR. (3) Results: The results obtained show that a "combination therapy" performed by combining the use of an anti-miR-10b-5p and a 1-(3',4',5'-trimethoxyphenyl)-2-aryl-1H-imidazole derivative is an encouraging strategy to boost the efficacy of anticancer therapies and at the same time to reduce side effects.
Assuntos
Antineoplásicos , Neoplasias Encefálicas , Glioblastoma , MicroRNAs , Antagomirs , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Imidazóis/farmacologia , MicroRNAs/metabolismoRESUMO
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene encodes for a chloride channel defective in Cystic Fibrosis (CF). Accordingly, upregulation of its expression might be relevant for the development of therapeutic protocols for CF. MicroRNAs are deeply involved in the CFTR regulation and their targeting with miRNA inhibitors (including those based on Peptide Nucleic Acids, PNAs)is associated with CFTR upregulation. Targeting of miR-145-5p, miR-101-3p, and miR-335-5p with antisense PNAs was found to be associated with CFTR upregulation. The main objective of this study was to verify whether combined treatments with the most active PNAs are associated with increased CFTR gene expression. The data obtained demonstrate that synergism of upregulation of CFTR production can be obtained by combined treatments of Calu-3 cells with antisense PNAs targeting CFTR-regulating microRNAs. In particular, highly effective combinations were found with PNAs targeting miR-145-5p and miR-101-3p. Content of mRNAs was analyzed by RT-qPCR, the CFTR production by Western blotting. Combined treatment with antagomiRNAs might lead to maximized upregulation of CFTR and should be considered in the development of protocols for CFTR activation in pathological conditions in which CFTR gene expression is lacking, such as Cystic Fibrosis.
Assuntos
Antagomirs , Fibrose Cística , MicroRNAs , Ácidos Nucleicos Peptídicos , Regiões 3' não Traduzidas , Antagomirs/farmacologia , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Humanos , MicroRNAs/genética , Ácidos Nucleicos Peptídicos/farmacologiaRESUMO
Glioblastoma multiforme (GBM) is a lethal malignant tumor accounting for 42% of the tumors of the central nervous system, the median survival being 15 months. At present, no curative treatment is available for GBM and new drugs and therapeutic protocols are urgently needed. In this context, combined therapy appears to be a very interesting approach. The isothiocyanate sulforaphane (SFN) has been previously shown to induce apoptosis and inhibit the growth and invasion of GBM cells. On the other hand, the microRNA miR-15b is involved in invasiveness and proliferation in GBM and its inhibition is associated with the induction of apoptosis. On the basis of these observations, the objective of the present study was to determine whether a combined treatment using SFN and a peptide nucleic acid interfering with miR-15b-5p (PNA-a15b) might be proposed for increasing the pro-apoptotic effects of the single agents. To verify this hypothesis, we have treated GMB U251 cells with SFN alone, PNA-a15b alone or their combination. The cell viability, apoptosis and combination index were, respectively, analyzed by calcein staining, annexin-V and caspase-3/7 assays, and RT-qPCR for genes involved in apoptosis. The efficacy of the PNA-a15b determined the miR-15b-5p content analyzed by RT-qPCR. The results obtained indicate that SFN and PNA-a15b synergistically act in inducing the apoptosis of U251 cells. Therefore, the PNA-a15b might be proposed in a "combo-therapy" associated with SFN. Overall, this study suggests the feasibility of using combined treatments based on PNAs targeting miRNA involved in GBM and nutraceuticals able to stimulate apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isotiocianatos/farmacologia , MicroRNAs/genética , Ácidos Nucleicos Peptídicos/farmacologia , Sulfóxidos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Glioblastoma , HumanosRESUMO
ß-thalassemias are among the most common inherited hemoglobinopathies worldwide and are the result of autosomal mutations in the gene encoding ß-globin, causing an absence or low-level production of adult hemoglobin (HbA). Induction of fetal hemoglobin (HbF) is considered to be of key importance for the development of therapeutic protocols for ß-thalassemia and novel HbF inducers need to be proposed for pre-clinical development. The main purpose on this study was to analyze Cinchona alkaloids (cinchonidine, quinidine and cinchonine) as natural HbF-inducing agents in human erythroid cells. The analytical methods employed were Reverse Transcription quantitative real-time PCR (RT-qPCR) (for quantification of γ-globin mRNA) and High Performance Liquid Chromatography (HPLC) (for analysis of the hemoglobin pattern). After an initial analysis using the K562 cell line as an experimental model system, showing induction of hemoglobin and γ-globin mRNA, we verified whether the two more active compounds, cinchonidine and quinidine, were able to induce HbF in erythroid progenitor cells isolated from ß-thalassemia patients. The data obtained demonstrate that cinchonidine and quinidine are potent inducers of γ-globin mRNA and HbF in erythroid progenitor cells isolated from nine ß-thalassemia patients. In addition, both compounds were found to synergize with the HbF inducer sirolimus for maximal production of HbF. The data obtained strongly indicate that these compounds deserve consideration in the development of pre-clinical approaches for therapeutic protocols of ß-thalassemia.
Assuntos
Alcaloides de Cinchona/farmacologia , Células Precursoras Eritroides/metabolismo , Hemoglobina Fetal/biossíntese , Talassemia beta/metabolismo , Células Precursoras Eritroides/patologia , Humanos , Células K562 , Talassemia beta/tratamento farmacológicoRESUMO
Peptide nucleic acids (PNAs) have been demonstrated to be very useful tools for gene regulation at different levels and with different mechanisms of action. In the last few years the use of PNAs for targeting microRNAs (anti-miRNA PNAs) has provided impressive advancements. In particular, targeting of microRNAs involved in the repression of the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis (CF), is a key step in the development of new types of treatment protocols. In addition to the anti-miRNA therapeutic strategy, inhibition of miRNA functions can be reached by masking the miRNA binding sites present within the 3'UTR region of the target mRNAs. The objective of this study was to design a PNA masking the binding site of the microRNA miR-145-5p present within the 3'UTR of the CFTR mRNA and to determine its activity in inhibiting miR-145-5p function, with particular focus on the expression of both CFTR mRNA and CFTR protein in Calu-3 cells. The results obtained support the concept that the PNA masking the miR-145-5p binding site of the CFTR mRNA is able to interfere with miR-145-5p biological functions, leading to both an increase of CFTR mRNA and CFTR protein content.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , MicroRNAs/metabolismo , Ácidos Nucleicos Peptídicos/metabolismo , Regiões 3' não Traduzidas/genética , Sítios de Ligação/genética , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , MicroRNAs/genéticaRESUMO
Two novel series of compounds based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and 4,5,6,7-tetrahydrobenzo[b]thiophene molecular skeleton, characterized by the presence of a 3',4',5'-trimethoxyanilino moiety and a cyano or an alkoxycarbonyl group at its 2- or 3-position, respectively, were designed, synthesized, and evaluated for antiproliferative activity on a panel of cancer cell lines and for selected highly active compounds, inhibition of tubulin polymerization, and cell cycle effects. We have identified the 2-(3',4',5'-trimethoxyanilino)-3-cyano-6-methoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine derivative 3a and its 6-ethoxycarbonyl homologue 3b as new antiproliferative agents that inhibit cancer cell growth with IC50 values ranging from 1.1 to 4.7 µM against a panel of three cancer cell lines. Their interaction with tubulin at micromolar levels leads to the accumulation of cells in the G2/M phase of the cell cycle and to an apoptotic cell death. The cell apoptosis study found that compounds 3a and 3b were very effective in the induction of apoptosis in a dose-dependent manner. These two derivatives did not induce cell death in normal human peripheral blood mononuclear cells, suggesting that they may be selective against cancer cells. Molecular docking studies confirmed that the inhibitory activity of these molecules on tubulin polymerization derived from binding to the colchicine site.
Assuntos
Antineoplásicos/química , Produtos Biológicos/química , Piridinas/química , Moduladores de Tubulina/química , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Produtos Biológicos/farmacologia , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colchicina/química , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Fase G2/efeitos dos fármacos , Células HeLa , Humanos , Células K562 , Leucócitos Mononucleares/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular/métodos , Piridinas/farmacologia , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Transfusão de Sangue Autóloga , Dopagem Esportivo , MicroRNAs/sangue , Medicina Esportiva , Adolescente , Adulto , Biomarcadores/sangue , Humanos , MasculinoRESUMO
There is a general agreement that pharmacologically mediated stimulation of human γ-globin gene expression and increase of production of fetal hemoglobin (HbF) is a potential therapeutic approach in the experimental therapy of ß-thalassemia and sickle cell anemia. Here, we report the development and characterization of cellular biosensors carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and ß-globin gene promoters, respectively; these dual-reporter cell lines are suitable to identify the induction ability of screened compounds on the transcription in erythroid cells of γ-globin and ß-globin genes by FACS with efficiency and reproducibility. Our experimental system allows to identify (a) HbF inducers stimulating to different extent the activity of the γ-globin gene promoter and (b) molecules that stimulate also the activity of the ß-globin gene promoter. A good correlation does exist between the results obtained by using the EGFP/RFP clones and experiments performed on erythroid precursor cells from ß-thalassemic patients, confirming that this experimental system can be employed for high-throughput screening (HTS) analysis. Finally, we have demonstrated that this dual-reporter cell line can be used for HTS in 384-well plate, in order to identify novel HbF inducers for the therapy of ß-thalassemia and sickle cell anemia. Graphical abstract.
Assuntos
Técnicas Biossensoriais , Diferenciação Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Regiões Promotoras Genéticas , Transcrição Gênica , Globinas beta/genética , gama-Globinas/genética , Eritrócitos/citologia , Hemoglobina Fetal/genética , Proteínas de Fluorescência Verde/genética , Humanos , Células K562 , Reprodutibilidade dos TestesRESUMO
Recent studies have identified and characterized a novel putative transcriptional repressor site in a 5' untranslated region of the Aγ-globin gene that interacts with the Ly-1 antibody reactive clone (LYAR) protein. LYAR binds the 5'-GGTTAT-3' site of the Aγ-globin gene, and this molecular interaction causes repression of gene transcription. In ß-thalassemia patients, a polymorphism has been demonstrated (the rs368698783 G>A polymorphism) within the 5'-GGTTAT-3' LYAR-binding site of the Aγ-globin gene. The major results gathered from surface plasmon resonance based biospecific interaction analysis (SPR-BIA) studies (using crude nuclear extracts, LYAR-enriched lysates, and recombinant LYAR) support the concept that the rs368698783 G>A polymorphism of the Aγ-globin gene attenuates the efficiency of LYAR binding to the LYAR-binding site. This conclusion was fully confirmed by a molecular docking analysis. This might lead to a very important difference in erythroid cells from ß-thalassemia patients in respect to basal and induced levels of production of fetal hemoglobin. The novelty of the reported SPR-BIA method is that it allows the characterization and validation of the altered binding of a key nuclear factor (LYAR) to mutated LYAR-binding sites. These results, in addition to theoretical implications, should be considered of interest in applied pharmacology studies as a basis for the screening of drugs able to inhibit LYAR-DNA interactions. This might lead to the identification of molecules facilitating induced increase of γ-globin gene expression and fetal hemoglobin production in erythroid cells, which is associated with possible reduction of the clinical severity of the ß-thalassemia phenotype. Graphical abstract.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Polimorfismo Genético , Ressonância de Plasmônio de Superfície/métodos , Talassemia beta/genética , gama-Globinas/genética , Sítios de Ligação , Células HEK293 , Humanos , Células K562 , Simulação de Acoplamento Molecular , Ligação Proteica , gama-Globinas/metabolismoRESUMO
BACKGROUND: Nonsense mutations promote premature translational termination, introducing stop codons within the coding region of mRNAs and causing inherited diseases, including thalassemia. For instance, in ß039 thalassemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, ribosomal read-through molecules, such as aminoglycoside antibiotics, have been tested on mRNAs carrying premature stop codons. These findings have introduced new hopes for the development of a pharmacological approach to the ß039 thalassemia therapy. While several strategies, designed to enhance translational read-through, have been reported to inhibit NMD efficiency concomitantly, experimental tools for systematic analysis of mammalian NMD inhibition by translational read-through are lacking. RESULTS: We developed a human cellular model of the ß039 thalassemia mutation with UPF-1 suppressed and showing a partial NMD suppression. CONCLUSIONS: This novel cellular model could be used for the screening of molecules exhibiting preferential read-through activity allowing a great rescue of the mutated transcripts.
Assuntos
RNA Helicases/genética , RNA Mensageiro/genética , Transativadores/genética , Talassemia beta/genética , Códon sem Sentido , Humanos , Células K562 , Degradação do RNAm Mediada por Códon sem Sentido , Mutação Puntual , Biossíntese de ProteínasRESUMO
Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorders.
Assuntos
Epigênese Genética , Histona Desacetilases/biossíntese , Debilidade Muscular/metabolismo , Miotonia Congênita/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/biossíntese , Animais , Histona Desacetilases/genética , Camundongos , Debilidade Muscular/genética , Debilidade Muscular/patologia , Mutação , Miotonia Congênita/genética , Miotonia Congênita/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genéticaRESUMO
BACKGROUND: Increase of the expression of γ-globin gene and high production of fetal hemoglobin (HbF) in ß-thalassemia patients is widely accepted as associated with a milder or even asymptomatic disease. The search for HbF-associated polymorphisms (such as the XmnI, BCL11A and MYB polymorphisms) has recently gained great attention, in order to stratify ß-thalassemia patients with respect to expectancy of the first transfusion, need for annual intake of blood, response to HbF inducers (the most studied of which is hydroxyurea). METHODS: Aγ-globin gene sequencing was performed on genomic DNA isolated from a total of 75 ß-thalassemia patients, including 31 ß039/ß039, 33 ß039/ß+IVSI-110, 9 ß+IVSI-110/ß+IVSI-110, one ß0IVSI-1/ß+IVSI-6 and one ß039/ß+IVSI-6. RESULTS: The results show that the rs368698783 polymorphism is present in ß-thalassemia patients in the 5'UTR sequence (+25) of the Aγ-globin gene, known to affect the LYAR (human homologue of mouse Ly-1 antibody reactive clone) binding site 5'-GGTTAT-3'. This Aγ(+25 G->A) polymorphism is associated with the Gγ-globin-XmnI polymorphism and both are linked with the ß039-globin gene, but not with the ß+IVSI-110-globin gene. In agreement with the expectation that this mutation alters the LYAR binding activity, we found that the Aγ(+25 G->A) and Gγ-globin-XmnI polymorphisms are associated with high HbF in erythroid precursor cells isolated from ß039/ß039 thalassemia patients. CONCLUSIONS: As a potential explanation of our findings, we hypothesize that in ß-thalassemia the Gγ-globin-XmnI/Aγ-globin-(G->A) genotype is frequently under genetic linkage with ß0-thalassemia mutations, but not with the ß+-thalassemia mutation here studied (i.e. ß+IVSI-110) and that this genetic combination has been selected within the population of ß0-thalassemia patients, due to functional association with high HbF. Here we describe the characterization of the rs368698783 (+25 G->A) polymorphism of the Aγ-globin gene associated in ß039 thalassemia patients with high HbF in erythroid precursor cells.
Assuntos
Hemoglobina Fetal/biossíntese , Polimorfismo Genético , Talassemia beta/genética , gama-Globinas/genética , Sítios de Ligação/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Desequilíbrio de Ligação , Masculino , Proteínas Nucleares/metabolismo , Mutação Puntual , Análise de Sequência de DNA , gama-Globinas/metabolismoRESUMO
The angelicin analogue 4,6,4'-trimethylangelicin (TMA) was recently reported as a strong inhibitor of nuclear factor-κB (NF-κB) activity and of the expression of the interleukin-8 (IL-8) gene in bronchial epithelial cells in which the inflammatory response has been challenged with P. aeruginosa, the most common bacterium found in the airways of patients affected by cystic fibrosis (CF). These findings encouraged us to analyze new synthetic analogues of TMA in order to evaluate their biological activities on human bronchial epithelial CF IB3-1 cells and to find more potent anti-NF-κB agents exhibiting only minor antiproliferative effects. Analogues able to inhibit NF-κB/DNA interaction at lower concentration than TMA were found and selected to investigate their biological activity on IB3-1 cells induced with TNF-α. In this biological system, NF-κB-mediated IL-8 gene expression was investigated. Some analogues showed similar activity to the lead compound TMA. Other analogues displayed higher activities; in particular, the most interesting compounds showing relevant anti-inflammatory effects were found to cause 56-83% reduction of IL-8 mRNA expression at low concentrations (1-10 µM), without changes in cell proliferation pattern, demonstrating their potential interest for a possible development of anti-inflammatory therapy of cystic fibrosis.
Assuntos
Fibrose Cística/metabolismo , Furocumarinas/química , Furocumarinas/farmacologia , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Linhagem Celular , Humanos , Interleucina-8/genética , Estrutura Molecular , NF-kappa B/genéticaRESUMO
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 ß-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 ß-thalassemia.