RESUMO
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
Although many potential applications in early clinical diagnosis have been proposed, the use of a surface plasmon resonance imaging (SPRI) technique for non-invasive prenatal diagnostic approaches based on maternal blood analysis is confined. Here, we report a nanoparticle-enhanced SPRI strategy for a non-invasive prenatal fetal sex determination based on the detection of a Y-chromosome specific sequence (single-gene SRY) in cell-free fetal DNA from maternal plasma. The SPR assay proposed here allows for detection of male DNA in mixtures of 2.5 aM male and female genomic DNAs with no preliminary amplification of the DNA target sequence, thus establishing an analytical protocol that does not require costly, time-consuming, and prone to sample contamination PCR-based procedures. Afterward, the developed protocol was successfully applied to reveal male cell-free fetal DNA in the plasma of pregnant women at different gestational ages, including early gestational ages. This approach would pave the way for the establishment of faster and cost-effective non-invasive prenatal testing.
Assuntos
Ácidos Nucleicos Livres , Nanopartículas , DNA/análise , Feminino , Humanos , Masculino , Gravidez , Análise para Determinação do Sexo/métodos , Ressonância de Plasmônio de SuperfícieRESUMO
Non-invasive prenatal testing (NIPT) is based on the detection and characterization of circulating cell-free fetal DNA (ccffDNA) in maternal plasma and aims to identify genetic abnormalities. At present, commercial NIPT kits can detect only aneuploidies, small deletions and insertions and some paternally inherited single-gene point mutations causing genetic diseases, but not maternally inherited ones. In this work, we have developed two NIPT assays, based on the innovative and sensitive droplet digital PCR (ddPCR) technology, to identify the two most common ß thalassemia mutations in the Mediterranean area (ß+IVSI-110 and ß039), maternally and/or paternally inherited, by fetal genotyping. The assays were optimized in terms of amplification efficiency and hybridization specificity, using mixtures of two genomic DNAs with different genotypes and percentages to simulate fetal and maternal circulating cell-free DNA (ccfDNA) at various gestational weeks. The two ddPCR assays were then applied to determine the fetal genotype from 52 maternal plasma samples at different gestational ages. The diagnostic outcomes were confirmed for all the samples by DNA sequencing. In the case of mutations inherited from the mother or from both parents, a precise dosage of normal and mutated alleles was required to determine the fetal genotype. In particular, we identified two diagnostic ranges for allelic ratio values statistically distinct and not overlapping, allowing correct fetal genotype determinations for almost all the analyzed samples. In conclusion, we have developed a simple and sensitive diagnostic tool, based on ddPCR, for the NIPT of ß+IVSI-110 and ß039 mutations paternally and, for the first time, maternally inherited, a tool, which may be applied to other single point mutations causing monogenic diseases.
Assuntos
Ácidos Nucleicos Livres , Talassemia beta , Ácidos Nucleicos Livres/genética , Feminino , Humanos , Mutação , Mutação Puntual , Reação em Cadeia da Polimerase , Gravidez , Diagnóstico Pré-Natal , Talassemia beta/genéticaRESUMO
The screening of chemical libraries based on cellular biosensors is a useful approach to identify new hits for novel therapeutic targets involved in rare genetic pathologies, such as ß-thalassemia and sickle cell disease. In particular, pharmacologically mediated stimulation of human γ-globin gene expression, and increase of fetal hemoglobin (HbF) production, have been suggested as potential therapeutic strategies for these hemoglobinopathies. In this article, we screened a small chemical library, constituted of 150 compounds, using the cellular biosensor K562.GR, 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. Then the identified compounds were analyzed as HbF inducers on primary cell cultures, obtained from ß-thalassemia patients, confirming their activity as HbF inducers, and suggesting these molecules as lead compounds for further chemical and biological investigations.
Assuntos
Anemia Falciforme/sangue , Descoberta de Drogas/métodos , Hemoglobina Fetal/biossíntese , Biossíntese de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Talassemia beta/sangue , Anemia Falciforme/tratamento farmacológico , Técnicas Biossensoriais/métodos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Humanos , Células K562 , Proteínas Luminescentes/genética , Bibliotecas de Moléculas Pequenas/uso terapêutico , Globinas beta/genética , Talassemia beta/tratamento farmacológico , gama-Globinas/genética , Proteína Vermelha FluorescenteRESUMO
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
Sickle Cell Disease (SCD) is a monogenic hereditary blood disorder caused by a single point mutation (ßS) in the ß globin gene resulting in an abnormal hemoglobin (HbS) that can polymerize within the erythrocytes, inducing their characteristic sickle shape. This causes hemolytic anemia and occlusive vessels for the most severe clinical status. Molecular analysis is crucial for fast and precise diagnosis of different forms of SCD, and, on the basis of underlying genotype, for supporting the most appropriate treatment options. In this context, we describe a simple and reproducible protocol for the molecular identification of the ßS mutation based on surface plasmon resonance (SPR) using the Biacore™ X100 affinity biosensor. This technology has already demonstrated its diagnostic suitability for the identification of point mutations responsible for genetic diseases such as cystic fibrosis and ß thalassemia, using a protocol based on immobilization of PCR products on the sensor chip. On the contrary, in this work we applied a SPR strategy based on an innovative interaction format, recently developed in our group also for ß thalassemia mutations. In particular, we correctly detected the ßS mutation responsible for SCD, both in homozygous and heterozygous states, after hybridization of two oligonucleotide probes (normal and mutated) for the ßS mutation, immobilized on sensor chip, with unbalanced PCR products obtained from 53 genomic DNAs carrying different ßS allele combinations.
RESUMO
BACKGROUND: Fetal sex determination is useful for families at risk of X-linked disorders, such as Duchenne muscular dystrophy, adrenal hypoplasia, hemophilia. At first, this could be obtained through invasive procedures such as amniocentesis and chorionic villus sampling, having a 1% risk of miscarriage. Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma, noninvasive prenatal testing permits the early diagnosis of fetal sex through analysis of cffDNA. However, the low amount of cffDNA relative to circulating maternal DNA requires highly sensitive molecular techniques in order to perform noninvasive prenatal diagnosis. In this context we employed droplet digital PCR (ddPCR) in order to evaluate the earliest possible fetal sex determination from circulating DNA extracted from plasma of pregnant women at different gestational ages. METHODS: We identified the fetal sex on cffDNA extracted from 29 maternal plasma samples at early gestational ages, several of them not suitable for qPCR determination, using ddPCR designed for SRY gene target. RESULTS: All maternal plasma samples were determined correctly for SRY gene target using ddPCR even at very early gestational age (prior to 7 weeks). CONCLUSIONS: The ddPCR is a robust, efficient and reliable technology for the earliest possible fetal sex determination from maternal plasma.
Assuntos
Ácidos Nucleicos Livres/sangue , Reação em Cadeia da Polimerase/métodos , Análise para Determinação do Sexo , Feminino , Feto , Idade Gestacional , Humanos , Masculino , GravidezRESUMO
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
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 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.
Assuntos
Proteínas de Transporte/genética , Redes Reguladoras de Genes , MicroRNAs/genética , Proteínas Nucleares/genética , gama-Globinas/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células Precursoras Eritroides/metabolismo , Humanos , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras , Talassemia beta/genética , gama-Globinas/metabolismoRESUMO
BACKGROUND: Cellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues. This approach is of great importance in studies on ß-thalassemia, since the recruitment of patients and collection of specimens can represent a crucial and often limiting factor in the experimental planning. METHODS: Erythroid precursor cells were obtained from 72 patients, mostly ß-thalassemic, expanded and cryopreserved. Expression of globin genes was analyzed by real time RT-qPCR. Hemoglobin production was studied by HPLC. RESULTS: In this paper we describe the production and validation of a Thal-Biobank constituted by expanded erythroid precursor cells from ß-thalassemia patients. The biobanked samples were validated for maintenance of their phenotype after (a) cell isolation from same patients during independent phlebotomies, (b) freezing step in different biobanked cryovials, (c) thawing step and analysis at different time points. Reproducibility was confirmed by shipping the frozen biobanked cells to different laboratories, where the cells were thawed, cultured and analyzed using the same standardized procedures. The biobanked cells were stratified on the basis of their baseline level of fetal hemoglobin production and exposed to fetal hemoglobin inducers. CONCLUSION: The use of biobanked cells allows stratification of the patients with respect to fetal hemoglobin production and can be used for determining the response to the fetal hemoglobin inducer hydroxyurea and to gene therapy protocols with reproducible results.
Assuntos
Bancos de Espécimes Biológicos , Talassemia beta/patologia , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Criopreservação , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/metabolismo , Eritropoetina/farmacologia , Hemoglobina Fetal/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Cinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma, diagnostic non-invasive prenatal methods have been developed or optimized for fetal sex determination and identification of genetic diseases. As far as fetal sex determination, this might be important for therapeutic intervention on sex-associated pathologies such as Duchenne muscular dystrophy, hemophilia and congenital adrenal hyperplasia. Surface plasmon resonance (SPR)-based biosensors might be useful for these studies, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses. METHODS: The Biacore™ X100 has been applied to identify Y-chromosome sequence in cffDNA obtained from plasma samples of 26 pregnant women at different gestational ages. We have performed SPR-based analysis of SRY PCR products using SRY-specific probes immobilized on the sensor chip. RESULTS: We have demonstrated that there is a statistically significant difference between samples collected by pregnancies carrying male or female fetuses. Moreover, cffDNA obtained at early gestational ages and not detectable by conventional quantitative real-time PCR can be discriminated with high accuracy and reliability using SPR-based biosensors. CONCLUSIONS: These data, in addition to their direct applicability in more extensive diagnostic trials, should be considered as the basis of future developments.
Assuntos
Cromossomos Humanos Y , Testes para Triagem do Soro Materno , Análise para Determinação do Sexo/métodos , Ressonância de Plasmônio de Superfície , Biomarcadores/sangue , Sistema Livre de Células , DNA/sangue , Feminino , Humanos , Masculino , Gravidez , Primeiro Trimestre da Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos TestesRESUMO
Rapamycin, an inhibitor of mTOR activity, is a potent inducer of erythroid differentiation and fetal hemoglobin production in ß-thalassemic patients. Mithramycin (MTH) was studied to see if this inducer of K562 differentiation also operates through inhibition of mTOR. We can conclude from the study that the mTOR pathway is among the major transcript classes affected by mithramycin-treatment in K562 cells and a sharp decrease of raptor protein production and p70S6 kinase is detectable in mithramycin treated K562 cells. The promoter sequence of the raptor gene contains several Sp1 binding sites which may explain its mechanism of action. We hypothesize that the G+C-selective DNA-binding drug mithramycin is able to interact with these sequences and to inhibit the binding of Sp1 to the raptor promoter due to the following results: (a) MTH strongly inhibits the interactions between Sp1 and Sp1-binding sites of the raptor promoter (studied by electrophoretic mobility shift assays, EMSA); (b) MTH strongly reduces the recruitment of Sp1 transcription factor to the raptor promoter in intact K562 cells (studied by chromatin immunoprecipitation experiments, ChIP); (c) Sp1 decoy oligonucleotides are able to specifically inhibit raptor mRNA accumulation in K562 cells. In conclusion, raptor gene expression is involved in mithramycin-mediated induction of erythroid differentiation of K562 cells and one of its mechanism of action is the inhibition of Sp1 binding to the raptor promoter.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Complexos Multiproteicos/antagonistas & inibidores , Plicamicina/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ciclo Celular , Diferenciação Celular , Expressão Gênica , Humanos , Células K562 , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Regiões Promotoras Genéticas , Ácido Pirrolidonocarboxílico/análogos & derivados , Ácido Pirrolidonocarboxílico/metabolismo , RNA Mensageiro/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Proteína Regulatória Associada a mTOR , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
MicroRNAs are a family of small noncoding RNAs regulating gene expression by sequence-selective mRNA targeting, leading to a translational repression or mRNA degradation. The oncomiR miR-221 is highly expressed in human gliomas, as confirmed in this study in samples of low and high grade gliomas, as well in the cell lines U251, U373 and T98G. In order to alter the biological functions of miR-221, a peptide nucleic acid targeting miR-221 (R8-PNA-a221) was produced, bearing a oligoarginine peptide (R8) to facilitate uptake by glioma cells. The effects of R8-PNA-a221 were analyzed in U251, U373 and T98G glioma cells and found to strongly inhibit miR-221. In addition, the effects of R8-PNA-a221 on p27(Kip1) (a target of miR-221) were analyzed in U251 and T98G cells by RT-qPCR and by Western blotting. No change of p27(Kip1) mRNA content occurs in U251 cells in the presence of PNA-a221 (lacking the R8 peptide), whereas significant increase of p27(Kip1) mRNA was observed with the R8-PNA-a221. These data were confirmed by Western blot assay. A clear increment of p27(Kip1) protein expression in the samples treated with R8-PNA-a221 was detected. In addition, R8-PNA-a221 was found able to increase TIMP3 expression (another target of miR-221) in T98G cells. These results suggest that PNAs against oncomiRNA miR-221 might be proposed for experimental treatment of human gliomas.
Assuntos
Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/metabolismo , MicroRNAs/metabolismo , Ácidos Nucleicos Peptídicos/farmacologia , Adulto , Análise de Variância , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Glioma/genética , Humanos , Masculino , MicroRNAs/genética , Modelos Moleculares , Fatores de TempoRESUMO
One of the most relevant pathophysiological hallmarks of ß-thalassemia is the accumulation of toxic α-globin chains inside erythroid cells, which is responsible for their premature death (hemolysis). In this context, the availability of an experimental model system mimicking the excess in α-globin chain production is still lacking. The objective of the present study was to produce and characterize K562 cellular clones forced to produce high amounts of α-globin, in order to develop an experimental model system suitable for studies aimed at the reduction of the accumulation of toxic α-globin aggregates. In the present study, we produced and characterized K562 cellular clones that, unlike the original K562 cell line, stably produced high levels of α-globin protein. As expected, the obtained clones had a tendency to undergo apoptosis that was proportional to the accumulation of α-globin, confirming the pivotal role of α-globin accumulation in damaging erythroid cells. Interestingly, the obtained clones seemed to trigger autophagy spontaneously, probably to overcome the accumulation/toxicity of the α-globin. We propose this new model system for the screening of pharmacological agents able to activate the full program of autophagy to reduce α-globin accumulation, but the model may be also suitable for new therapeutical approaches targeted at the reduction of the expression of the α-globin gene.
Assuntos
Autofagia , alfa-Globinas , Humanos , alfa-Globinas/biossíntese , alfa-Globinas/genética , Autofagia/genética , Biomarcadores , Células Clonais , Células K562RESUMO
Gene therapy might fall short in achieving a complete reversion of the ß-thalassemic phenotype due to current limitations in vector design and myeloablative regimen. Following gene transfer, all or a large proportion of erythroid cells might express suboptimal levels of ß-globin, impairing the therapeutic potential of the treatment. Our aim was to evaluate whether, in absence of complete reversion of the ß-globin phenotype upon gene transfer, it is possible to use fetal hemoglobin induction to eliminate the residual α-globin aggregates and achieve normal levels of hemoglobin. Transgenic K562 cell lines and erythroid precursor cells from ß(0)39-thalassemia patients were employed. Gene therapy was performed with the lentiviral vector T9W. Induction of fetal hemoglobin was obtained using mithramycin. Levels of mRNA and hemoglobins were determined by qRT-PCR and HPLC. First, we analyzed the effect of mithramycin on K562 transgenic cell lines harboring different copies of a lentiviral vector carrying the human ß-globin gene, showing that γ-globin mRNA expression and HbF production can be induced in the presence of high levels of ß-globin gene expression and HbA accumulation. We then treated erythroid progenitor cells from ß-thalassemic patients with T9W, which expresses the human ß-globin gene and mithramycin separately or in combination. When transduction with our lentiviral vector is insufficient to completely eliminate the unpaired α-globin chains, combination of ß-globin gene transfer therapy together with fetal hemoglobin induction might be very efficacious to remove the excess of α-globin proteins in thalassemic erythroid progenitor cells.
Assuntos
Células Precursoras Eritroides/efeitos dos fármacos , Hemoglobina Fetal/metabolismo , Terapia Genética , Hemoglobina A/genética , Plicamicina/uso terapêutico , Talassemia beta/terapia , Adulto , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/uso terapêutico , Células Cultivadas , Terapia Combinada/métodos , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/fisiologia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Células HEK293 , Hemoglobina A/metabolismo , Humanos , Células K562 , Plicamicina/farmacologia , Globinas beta/genética , Talassemia beta/genética , Talassemia beta/metabolismoRESUMO
The pandemic caused by the SARS-CoV-2 virus (COVID-19) is still a major health issue. The COVID-19 pandemic has forced the university teaching to consider in high priority the switch from in-presence teaching to remote teaching, including laboratory teaching. While excellent virtual-laboratory teaching has been proposed and turned out to be very useful, the need of a real-laboratory in-presence teaching is still a major need. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. SARS-CoV-2 diagnostics, and (b) on technologies that are playing a central role in applied biochemistry and molecular biology, i.e. PCR and RT-PCR. The aims of the practical laboratory were to determine: (a) the possibility to identify SARS-CoV-2 sequences starting from a recombinant plasmid and (b) the possibility to discriminate cells with respect to the expression of SARS-CoV-2 Spike protein. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). We suggest that this laboratory practical exercises should be considered for face-to-face teaching especially if the emergency related to the COVID-19 pandemic is maintained. The teaching protocol here described might be considered in order to perform fast but meaningful in-presence teaching, making feasible the division of crowded classes in low-number cohorts of students, allowing the maintenance of the required social distance.
Assuntos
Bioquímica , Farmacologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Ensino , Bioquímica/educação , Farmacologia/educação , RNA , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Introduction: ß-thalassemia is caused by autosomal mutations in the ß-globin gene, which induce the absence or low-level synthesis of ß-globin in erythroid cells. It is widely accepted that a high production of fetal hemoglobin (HbF) is beneficial for patients with ß-thalassemia. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus that serves as a strong HbF inducer in vitro and in vivo. In this study, we report biochemical, molecular, and clinical results of a sirolimus-based NCT03877809 clinical trial (a personalized medicine approach for ß-thalassemia transfusion-dependent patients: testing sirolimus in a first pilot clinical trial, Sirthalaclin). Methods: Accumulation of γ-globin mRNA was analyzed using reverse-transcription quantitative polymerase chain reaction (PCR), while the hemoglobin pattern was analyzed using high-performance liquid chromatography (HPLC). The immunophenotype was analyzed using a fluorescence-activated cell sorter (FACS), with antibodies against CD3, CD4, CD8, CD14, CD19, CD25 (for analysis of peripheral blood mononuclear cells), or CD71 and CD235a (for analysis of in vitro cultured erythroid precursors). Results: The results were obtained in eight patients with the ß+/ß+ and ß+/ß0 genotypes, who were treated with a starting dosage of 1 mg/day sirolimus for 24-48 weeks. The first finding of this study was that the expression of γ-globin mRNA increased in the blood and erythroid precursor cells isolated from ß-thalassemia patients treated with low-dose sirolimus. This trial also led to the important finding that sirolimus influences erythropoiesis and reduces biochemical markers associated with ineffective erythropoiesis (excess free α-globin chains, bilirubin, soluble transferrin receptor, and ferritin). A decrease in the transfusion demand index was observed in most (7/8) of the patients. The drug was well tolerated, with minor effects on the immunophenotype, and an only side effect of frequently occurring stomatitis. Conclusion: The data obtained indicate that low doses of sirolimus modify hematopoiesis and induce increased expression of γ-globin genes in a subset of patients with ß-thalassemia. Further clinical trials are warranted, possibly including testing of the drug in patients with less severe forms of the disease and exploring combination therapies.
RESUMO
Ultrasensitive detection protocols not requiring polymerase chain reaction (PCR)-mediated target DNA amplification are expected to significantly improve our possibilities in several research and diagnostic applications for which minute cell quantities are available. For this reason we have tested a nanoparticle-enhanced surface plasmon resonance imaging (SPRI) sensing strategy to detect point mutations in nonamplified genomic DNA. We have used genomic DNAs, not subject to costly, time-consuming, and prone to contamination PCR-based amplification procedures, obtained from both healthy individuals and homozygous or heterozygous patients affected by ß-thalassemia, in order to demonstrate the specificity and the sensitivity of the described sensing strategy. The assay we describe is ultrasensitive and convenient. Attomolar concentrations of target genomic DNA are detected, DNAs from healthy individuals and homozygous or heterozygous patients affected by ß-thalassemia are discriminated, and only simple manipulations of the genetic samples are required before the analysis. The proposed ultrasensitive detection of DNA point mutations involved in genomic disorders possibly represents an important advantage in several biomedical applications.