RESUMO
The XmnI Gg -158 C/T polymorphism has been widely associated with fetal hemoglobin (HbF) levels, the severity of disease, and the response to the drug hydroxyurea (HU) in both ß-thalassemia (ß-thal) and sickle cell disease (SCD) patients. However, the functional significance of this single nucleotide polymorphism (SNP) remains unclear. To gain insight, green fluorescence protein (GFP) cassettes harboring the XmnI C or T alleles in their left homology arms (i.e. Gg promoters) were knocked into the Gg gene(s) of K562 cells via CRISPR/Cas9. Subsequently, the GFP fluorescence levels were compared in the ensuing cell populations and isolated clones. In both instances, median fluorescence intensities (MFI) of the knockin cells having the inserted XmnI T allele were higher than those having the XmnI C allele. Our results suggest that the XmnI T allele can increase Gg expression in K562 cells. The possible functional significance of the XmnI Gg -158 C/T polymorphism provides a rationale for the aforementioned associations. Furthermore, the XmnI polymorphism as a functional SNP substantiates its importance as a prognostic marker.
Assuntos
Alelos , Anemia Falciforme , Sistemas CRISPR-Cas , Edição de Genes , Polimorfismo de Nucleotídeo Único , Talassemia beta , Humanos , Talassemia beta/genética , Células K562 , Edição de Genes/métodos , Anemia Falciforme/genética , Sistemas CRISPR-Cas/genética , Polimorfismo de Nucleotídeo Único/genética , Marcadores Genéticos/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Sequência de BasesRESUMO
The CRISPR/Cas9 system is a rapid, simple, and often extremely efficient gene editing method. This method has been used in a variety of organisms and cell types over the past several years. However, using this technology for generating gene-edited animals involves a number of obstacles. One such obstacle is mosaicism, which is common in founder animals. This is especially the case when the CRISPR/Cas9 system is used in embryos. Here we review the pros and cons of mosaic mutations of gene-edited animals caused by using the CRISPR/Cas9 system in embryos. Furthermore, we will discuss the mechanisms underlying mosaic mutations resulting from the CRISPR/Cas9 system, as well as the possible strategies for reducing mosaicism. By developing ways to overcome mosaic mutations when using CRISPR/Cas9, genotyping for germline gene disruptions should become more reliable. This achievement will pave the way for using the CRISPR technology in the research and clinical applications where mosaicism is an issue.
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Sistemas CRISPR-Cas , Edição de Genes/métodos , Mosaicismo , Animais , Animais Geneticamente Modificados , Feminino , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Humanos , Masculino , Modelos Genéticos , Mutação , GravidezRESUMO
OBJECTIVE: To knock-in an EGFP cassette into the γ-globin genes of K562 cells via CRISPR/Cas9, and to assess expression and hydroxyurea (HU)-mediated induction of the targeted EGFP transgene. RESULTS: The EGFP cassettes were specifically knocked into the Gγ gene. EGFP expression was detected in the targeted cell population and isolated clones. Furthermore, EGFP transcript and fluorescence levels were significantly induced following HU-treatment. CONCLUSION: This system is readily utilizable for genome scale studies of cis-acting regulatory elements which are implicated in γ-globin expression or HU-mediated induction.
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Engenharia Celular/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Introdução de Genes/métodos , Proteínas de Fluorescência Verde/biossíntese , Hidroxiureia/metabolismo , Proteínas Recombinantes/biossíntese , Ativação Transcricional , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Fluorescência Verde/genética , Humanos , Células K562 , Proteínas Recombinantes/genética , gama-Globinas/genéticaRESUMO
B-cell lymphoma 2 (BCL2) family proteins play a critical role in tuning cell death processes. Almost in half of all human cancers, a dysregulation in BCL2 family gene expression has been shown which made it an impressive target for human gene therapy as a novel approach in cancers. In this study we will optimize lentiviral-mediated RNA interference (RNAi), recombinant lentiviruses accommodating anti-BCL2 micro adaptive short hairpin RNAs (shRNAs), to downregulate BCL2 in human embryonic kidney 293T (HEK293T) cells to produce stable cell lines. We tested 4 different Dharmacon™ GIPZ™ shRNAmir lentiviral vectors targeting BCL2 in different positions and a pGIPZ non-silencing shRNAmir lentiviral vector (as a negative control). Lentivirus packaging was performed by the calcium phosphate precipitation method. HEK293T cells were transduced by each type of recombinant lentiviruses individually and selected by puromycin within 10 days. The relative mRNA level and protein expression were assayed by using real-time polymerase chain reaction (PCR) technic and western blotting, respectively. Lentivirus (LV) packaging was performed in high efficiency (transfection rate was > 90%). Recombinant viruses of 4 expression vector addition to a control vector were produced then transduced to HEK293T cells successfully. All the 4 cell groups showed a significant down regulation of BCL2 gene (~90-95%) at mRNA level compared to the control group (p<0.01) but differences between silenced groups were not significant (P > 0.05). We showed that the lentivirus-mediated RNAi technique is an efficient method to establish HEK293 cell lines with stable down-regulation of BCL2 gene.
Assuntos
Técnicas de Silenciamento de Genes/métodos , Lentivirus/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Interferente Pequeno/genética , Western Blotting , Células HEK293 , Humanos , Interferência de RNARESUMO
The Krüppel-like factor 1 (KLF1) is an essential erythroid-specific transcription factor. Mutations in the human KLF1 gene have different phenotypic effects, ranging from increased Hb F levels to the disruption of erythropoiesis. Here, we screened 227 Iranian ß-thalassemia (ß-thal) patients for the presence of KLF1 mutations by using the single-strand conformational polymorphism (SSCP) approach. Our aim was to assess the potential effect of these mutations on the ß-thal disease severity. After screening, two variants were found. One patient carried a potentially deleterious variant (Polyphen-2) in exon 2 (p.F182L). Another patient was homozygous for a previously unreported intronic variant (KLF1: c.911 + 84A > G). The patient with the p.F182L variant (KLF1: c.544T > C) had noticeably high Hb A2 levels (7.6%), consistent with the phenotypic effect of several previously characterized KLF1 mutations in the same exonic region. In addition, he had higher platelet counts (1,069,000/µL) compared to other patients in the cohort.
Assuntos
Fatores de Transcrição Kruppel-Like/genética , Polimorfismo Genético , Talassemia beta/genética , Adulto , Sequência de Bases , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Hemoglobina A2/análise , Humanos , Irã (Geográfico)/epidemiologia , Masculino , Mutação , Adulto Jovem , Talassemia beta/sangue , Talassemia beta/epidemiologiaRESUMO
Reactivation of the fetal hemoglobin (HbF) in adult erythroid cells via genome editing is a strategy for the treatment of ß-thalassemia and sickle cell disease. In related reports, the reactivation of HbF is regularly examined in erythroblasts which are generated from the adult CD34+ hematopoietic stem and progenitor cells (HSPCs). However, the procurement of adult HSPCs, either from the bone-marrow (BM) or from mobilized peripheral-blood (mPB), is difficult. Cord-blood (CB) is a readily available source of HSPCs. CB-HSPCs, however, produce high quantities of HbF following differentiation into the erythroid lineage-a potential drawback in such studies. Here, we have edited the BCL11A enhancer (a well-characterized HbF-quantitative trait loci or QTL) via CRISPR/Cas9 in order to determine whether HbF reactivation could be detected in CB-HSPC-derived erythroblasts. In the edited erythroblasts, insertion/deletion (indel) frequencies of 74.0-80.4% and BCL11A RNA reduction levels of 92.6 ± 5.1% (P < 0.0001) were obtained. In turn, the γ/ß-globin transcript ratios were increased from 11.3 ± 1.1-fold to 77.1 ± 2.0-fold, i.e., by 6.8-fold (P < 0.0001)-and the HbF% levels increased from 34.3% in the control population to 43.5% in the BCL11A edited erythroblasts. Our results suggest that γ-globin/HbF reactivation via genome editing can be detected in CB-HSPCs generated erythroblasts-rendering CB-HSPCs a useful model for similar studies.
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Hydroxyurea (HU) is a drug that induces fetal hemoglobin production. As a result, HU is widely used to treat ß-thalassemia (ß-thal) patients. However, the response of these patients to HU varies. Some ß-thal patients respond favorably to treatment while others do not respond at all. HU has a number of side-effects and therefore its targeted prescription is beneficial. Hence, identifying the genetic determinants which lead to the differential HU response is important. This review summarizes recent findings which have shed light on this topic. Special emphasis is given to the mechanisms and genetic loci which may govern these differences. These findings have helped identify several single nucleotide polymorphisms which associate with the response to HU in both ß-thal and sickle cell disease patients.
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Hidroxiureia/uso terapêutico , Talassemia beta/tratamento farmacológico , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Hidroxiureia/química , Hidroxiureia/farmacologia , Polimorfismo de Nucleotídeo Único/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Resultado do Tratamento , Talassemia beta/genética , Talassemia beta/metabolismoRESUMO
Recently, five genetic modifiers [ß-globin mutations, coinheritance of α-thalassemia (α-thal), XmnI polymorphism and single nucleotide polymorphisms (SNPs) in the BCL11A and HBS1L-MYB loci] were used to predict the ß-thal major (ß-TM) or ß-thal intermedia (ß-TI) types in 106 French patients with 83.2% accuracy. The dichotomous grouping was based on the age when the patient received his/her first transfusion (4 years). Here, a similar study was conducted in a cohort of 306 Iranian ß-thal patients having distinct ß-globin mutations and minor allele frequencies of key SNPs in these loci. Multivariate regression analyses and a simple scoring system were used to predict the ß-TM/ß-TI types using three scenarios: 1) when considering only the severe ß-TM and the mild ß-TI cases, 2) using clinical parameters for ß-thal typing, and 3) using age at first transfusion as the basis for classification. Using these scenarios, the ß-thal types could be correctly predicted in 77.6, 75.5 and 68.0% of cases, respectively.
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Análise Multivariada , Análise de Regressão , Talassemia beta/diagnóstico , Talassemia beta/genética , Adulto , Sítios de Ligação/genética , Proteínas de Transporte/genética , Estudos de Coortes , DNA Intergênico/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Feminino , Proteínas de Ligação ao GTP/genética , Frequência do Gene , Genótipo , Proteínas de Choque Térmico HSP70/genética , Humanos , Irã (Geográfico) , Masculino , Mutação , Proteínas Nucleares/genética , Fatores de Alongamento de Peptídeos/genética , Polimorfismo de Nucleotídeo Único , Prognóstico , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Repressoras , Sensibilidade e Especificidade , Adulto Jovem , Globinas beta/genética , Talassemia beta/classificaçãoRESUMO
1. Hydroxyurea (HU) is a drug used for the treatment of haemoglobinopathies. Hydroxyurea functions by upregulating γ-globin transcription and fetal haemoglobin (HbF) production in erythroid cells. The K562 erythroleukaemia cell line is widely used as a model system in which to study the mechanism of γ-globin induction by HU. However, the transcription factors required for the upregulation of γ-globin expression by HU in K562 cells have not been identified. Similarities between the HU and sodium butyrate (SB) pathways suggest cAMP response element-binding protein (CREB) 1 as a potential candidate. Thus, the aim of the present study was to investigate the possible role of CREB1 in the HU pathway. 2. Experiments were performed using transient and stable RNA interference (RNAi) to show that CREB1 is necessary for HU-mediated induction of γ-globin expression and haemoglobin production in K562 cells. 3. Furthermore, western blot analyses demonstrated that CREB1 becomes phosphorylated in a dose-dependent manner after HU (100-400 µmol/L) treatment of K562 cells for 72 h. 4. We also investigated role of a Gγ promoter CREB1 response element (G-CRE) in this pathway. Quantitative amplification refractory mutation system-polymerase chain reaction experiments were performed to demonstrate that HU induces the expression of both Gγ and Aγ in this cell line. In addition, electrophoretic mobility shift assays were used to show that levels of CREB1 complexes binding to the G-CRE site are increased following HU treatment and are decreased in CREB1-knockdown cells. 5. The results suggest that CREB1 is necessary for γ-globin induction by HU in K562 cells, a role that may be mediated, in part, through the G-CRE element.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Regulação Neoplásica da Expressão Gênica , Hidroxiureia/farmacologia , gama-Globinas/biossíntese , gama-Globinas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Hemoglobinas/biossíntese , Humanos , Células K562 , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Hydroxyurea (HU), a drug which can reactivate fetal hemoglobin (Hb F) production, is frequently prescribed to ß-thalassemia (ß-thal) patients. However, transfusion requirements of only a subset of patients are reduced upon HU treatment. Because of its potential side-effects, targeted prescription of HU is imperative. To identify genetic markers that correlate with drug response, we have carried out a retrospective association study of single nucleotide polymorphisms (SNPs) in three Hb F quantitative trait loci (QTLs): the XmnI polymorphism, BCL11A, and the HBS1L-MYB intergenic region, with the response to HU in a cohort of 81 transfusion-dependent Iranian ß-thal patients. An increase in blood transfusion intervals post-therapy was used to measure drug response. Our results suggest that presence of the XmnI T/T genotype or the BCL11A rs766432 C allele correlates strongly with response to HU (p <0.001). Accordingly, these markers may be used to accurately predict the HU response of Iranian ß-thal patients.
Assuntos
Hemoglobina Fetal/genética , Hidroxiureia/uso terapêutico , Polimorfismo de Nucleotídeo Único , Talassemia beta/tratamento farmacológico , Talassemia beta/genética , Adulto , Antidrepanocíticos/uso terapêutico , Transfusão de Sangue , Feminino , Frequência do Gene , Genótipo , Humanos , Irã (Geográfico) , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Resultado do Tratamento , Adulto Jovem , Talassemia beta/terapiaRESUMO
BACKGROUND: The PX330 and the related PX459 plasmids are widely used for Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-mediated genome editing. Screening for plasmids containing the correct sgRNA template insertion is one of the most important steps in this system. Different methods for screening the sgRNA inserts have been deployed. One such method is Restriction Enzyme (RE) mapping. Restriction enzyme mapping can be used to screen for numerous plasmid recombinants simultaneously. METHODS: In this study, the sgRNA templates were initially cloned into the above PX459 plasmids. Subsequently, the accuracy of the constructs was determined by RE mapping. RESULTS: This method was established to screen for sgRNA-bearing PX459 plasmids. However, numerous anomalies were detected after ligation of sgRNA templates into RE digested PX459 plasmids. CONCLUSION: Our data suggest that RE mapping is only appropriate as an initial screen and that the identity of all plasmids with the correctly identified RE maps should be confirmed by Sanger sequencing.
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Since its inception, the CRISPR/Cas9 technology has been widely utilized for the targeted insertion of donor DNAs into mammalian genomes. A shortcoming with the earlier knock-in (KI) approaches, however, has been the low efficiency of targeted integrations-especially in primary cells and mouse embryos. Since, a variety of novel strategies have been developed towards improving the KI efficiencies in select target cells. In this review, the current applications of CRISPR/Cas9-mediated KIs in mammalian cells are described. Furthermore, the recent strategies which have been developed in order to augment the CRISPR/Cas9-mediated KI efficiencies are summarized.
Assuntos
Sistemas CRISPR-Cas , Técnicas de Introdução de Genes/métodos , Mamíferos/genética , Animais , Edição de Genes , Humanos , CamundongosRESUMO
BACKGROUND: The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRIS-PR) and CRISPR-associated protein (Cas) system has been used as a powerful tool for genome engineering. In this study, the application of this system is reported for targeting Rag genes to produce mutant mouse NIH/3T3 cell line. The Rag1 and Rag2 genes are essential for generation of mature B and T lymphocytes. Disruption of Rag genes causes disease like Severe Combined Immunodeficiency syndrome (SCID). Here, the efficiency and specificity of CRISPR system were tested with highly active sgRNAs to generate novel mutations in the NIH/3T3 mouse cell line. METHODS: Four single guide RNAs were designed to target sequences in the coding region of the Rag1 and Rag2 genes. Four sgRNA-CAS9 plasmids were tested to target Rag1 and Rag2. RESULTS: Based on T7 endonuclease assay and sequencing analysis, the expression of sgRNAs targeting two sites in Rag1 resulted in deletion of the intervening DNA fragment. The expression of sgRNAs with Cas9 targeting two sites in Rag2 gene resulted in indel mutations at both sites. In this report, fragment deletion in Rag1 gene was detected in about 50% of transfected cells. CONCLUSION: Therefore, CRISPR/Cas9 system can be highly efficient and specific when gRNAs are designed rationally and provides a powerful approach for genetic engineering of cells and model animals.
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BACKGROUND: Recombination Activating Genes (RAG) mutated embryonic stem cells are (ES) cells which are unable to perform V (D) J recombination. These cells can be used for generation of immunodeficient mouse. Creating biallelic mutations by CRISPR/Cas9 genome editing has emerged as a powerful technique to generate site-specific mutations in different sequences. OBJECTIVES: The main purposes of this study were to achieve complete knock-out of RAG1 gene by investigating the nature of mutations in mutant mESC and to generate RAG1 knock-out mESCs containing homozygous indels with the aim of creating desired and specific RAG-1 -/- mutant mouse in a shorter period of time. MATERIALS AND METHODS: Here, we first utilized CRISPR/Cas9 system to target RAG1/RAG2 genes in NIH3T3 cells to test the activity and efficiency of our CRISPR system. Then we used the system for targeting RAG1 gene in mouse embryonic stem cell (mESCs) to generate knock-out embryonic stem cells. This method combined with highly active single guide RNA (sgRNA) is an efficient way to produce new RAG1-knockout mESCs in the selected regions of early coding DNA sequence, approximately between nucleotide c. 512-c. 513 and nucleotide c. 725-c. 726 of RAG1 coding sequence that had not been targeted previously. RESULTS: CRISPR gene editing resulted in a multitude of engineered homozygous and compound heterozygous mutations, including both in-frame and out-of-frame indels in 92% of mES cell clones. Most of the mutations generated by CRISPR/Cas9 system were out-of-frame, resulting in a complete gene knockout. In addition, 59% of the mutant ES cell clones carried out-of-frame homozygous indel mutations. The RAG1-knockout mESC clones retained normal morphology and pluripotent gene expression. CONCLUSIONS: Our study demonstrated that CRISPR/Cas9 system can efficiently create biallelic indels containing both homozygous and compound heterozygous RAG1 mutations in about 92% of the mutant mESC clones. The 59% of mutant ES cell clones carried out-of-frame homozygous indel mutations.
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AIM: To identify the BCL11A intron-2 enhancer linkage disequilibrium (LD) block, harboring two previously identified SNPs, associating with the hydroxyurea response in ß-thalassemia patients and the functional significance of this region. MATERIALS & METHODS: Several neighboring SNPs were genotyped in our cohort. The associating LD block was identified, and its function studied in K562 erythroid cells via CRISPR/Cas9 genome editing. RESULTS: A haplotype harboring three tag SNPs correlated significantly with the HU-response and BCL11A transcript levels in the patients' reticulocytes. Two deletions encompassing this LD block significantly reduced BCL11A transcript levels in K562 cells. CONCLUSION: Our data suggest an essential role for this LD block in BCL11A expression levels and the response to hydroxyurea in ß-thalassemia patients.
Assuntos
Proteínas de Transporte/genética , Hidroxiureia/uso terapêutico , Proteínas Nucleares/genética , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Talassemia beta/tratamento farmacológico , Estudos de Coortes , Elementos Facilitadores Genéticos , Frequência do Gene , Genótipo , Células HEK293 , Haplótipos , Humanos , Hidroxiureia/administração & dosagem , Hidroxiureia/farmacocinética , Células K562 , Desequilíbrio de Ligação , Proteínas Repressoras , Reticulócitos/metabolismo , Transfecção , Talassemia beta/sangue , Talassemia beta/genéticaRESUMO
A majority of acute lymphoblastic leukemia patients overexpress CREB in the bone marrow. However, the functional significance of this up-regulation and the detailed molecular mechanism behind the regulatory effect of CREB on the growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells has not been elucidated. We demonstrated here that CREB knockdown induced apoptosis and impaired growth of BCP-ALL NALM-6 cells which was associated with caspase activation. The gene expression levels of prosurvival signals Bcl-2, Mcl-1, Bcl-xL, survivin and XIAP were down-regulated upon CREB suppression. These findings indicate a critical role for CREB in proliferation, survival, and apoptosis of BCP-ALL cells. The data also suggest that CREB could possibly serve as potential therapeutic target in BCP-ALL.
Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Sobrevivência Celular/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Possible mechanisms of Alzheimer Disease (AD) such as inflammation and oxidative stresses in the brain led us to investigate potential AD therapeutics of Melilotus officinalis, an herbal extract, with possible role as an anti-inflammatory and anti-oxidant agent. Among different genes which had important role in Sporadic AD (SAD), three genes including DAXX, NFkB and VEGF have shown significant statistical diversity in the brains of Alzheimer patients. METHODS: These genes were chosen to be investigated for neuroprotective effects of the extract by comparing the expression level in the hippocampus of Sporadic AD (SAD) rat model using quantitative polymerase chain reaction (qPCR) in the treated and untreated groups. In addition, therapeutic effects at the behavioral, learning and memory level by Morris Water Maze (MWM) test were investigated. RESULTS: The results represented significant decreased expression in Daxx, Nfkb and Vegf genes in the SAD rat's model treated with the herbal extract compared to the Streptozotocin-induced (STZ-induced) rats. Furthermore, no significant changes were seen in swimming distance and time for finding the hidden platform in the herbal-treated compared to the STZ-induced group. In memory level, no significant changes were observed among treated and untreated groups. CONCLUSION: It seems that the herbal extract may have significant effect on Alzheimer-related gene expression changes but not on clinical levels.
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Purpose: Sporadic Alzheimer's disease (AD) accounts for over 95% of cases. Possible mechanisms of AD such as inflammation and oxidative stresses in the brain motivate researchers to follow many therapies which would be effective, especially in the early stages of the disease. IMOD, the herbal extract of R. Canina, T. Vulgare and U. Dioica plant species enriched with selenium, has anti-inflammatory, immunoregulatory and protective effects against oxidative stress. Methods: In this study three AD-related genes, DAXX, NFκß and VEGF, were chosen as candidate to investigate the neuroprotective effect of the extract by comparing their expression levels in the hippocampus of rat model of sporadic AD, using qPCR in the herbal-treated and control groups. The therapeutic effects on learning and memory levels were evaluated by Morris Water Maze (MWM) test. Results: Gene expression results were indicative of significant up-regulation of Vegf in rat's hippocampus after treatment with the herbal extract comparing to model group (P-value= 0.001). The MWM results showed significant changes in path length and time for finding the hidden platform in all groups during test and the same change in the treated comparing to the control group in memory level. Conclusion: It could be concluded that the herbal extract may have significant effect on gene expression but not on behavioral level.
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Purpose: Alzheimer's disease (AD) is pathologically defined by the presence of amyloid plaques and tangles in the brain, therefore, any drug or compound with potential effect on lowering amyloid plaques, could be noticed for AD management especially in the primary phases of the disease. Ectoine constitutes a group of small molecule chaperones (SMCs). SMCs inhibit proteins and other changeable macromolecular structures misfolding from environmental stresses. Ectoine has been reported successfully prohibit insulin amyloid formation in vitro. Methods: We selected eight genes, DAXX, NFκß, VEGF, PSEN1, MTAP2, SYP, MAPK3 and TNFα genes which had previously showed significant differential expression in Alzheimer human brain and STZ- rat model. We considered the neuroprotective efficacy by comparing the expression of candidate genes levels in the hippocampus of rat model of Sopradic Alzheimer's disease (SAD), using qPCR in compound-treated and control groups as well as therapeutic effects at learning and memory levels by using Morris Water Maze (MWM) test. Results: Our results showed significant down-regulation of Syp, Mapk3 and Tnfα and up-regulation of Vegf in rat's hippocampus after treatment with ectoine comparing to the STZ-induced group. In MWM, there was no significant change in swimming distance and time for finding the hidden platform in treated comparing to STZ-induced group. In addition, it wasn't seen significant change in compound-treated comparing to STZ-induced and control groups in memory level. Conclusion: It seems this compound may have significant effect on expression level of some AD- related genes but not on clinical levels.
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Elevated cAMP levels in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells attenuate the doxorubicin-induced p53 accumulation and protect cells against apoptosis. cAMP responsive element binding protein (CREB) is a cAMP-stimulated transcription factor that regulates genes whose deregulated expression cooperate in oncogenesis. In the present study, we investigated the role of CREB on inhibitory effect of cAMP on apoptosis and p53 accumulation in BCP-ALL NALM-6 cells. To determine whether targeting CREB can modulate the effect of cAMP on doxorubicin-induced apoptosis, we knocked down CREB in NALM-6 cells using lentiviral CREB shRNA. Knocked down cells were treated with doxorubicin in the presence or absence of cAMP-elevating agents. p53 protein level and apoptosis were assessed by western blot analysis and flow cytometry, respectively. p53 protein expression was reduced in cells treated with combination of cAMP-elevating agents and doxorubicin in contrast to cells treated with doxorubicin alone even in CREB-knocked down cells. Apoptosis assay showed that the cAMP-elevating agents decreased doxorubicin-induced apoptosis in CREB-knocked down and control cells. Although, CREB plays a particularly important role in cAMP signaling pathway our data suggest that CREB does not mediate the inhibitory effect of cAMP on doxorubicin-induced apoptosis and p53 accumulation in BCP-ALL NALM-6 cells.