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BACKGROUND: High-throughput experiments provide deep insight into the molecular biology of different species, but more tools need to be developed to handle this type of data. At the transcriptomics level, quantitative Polymerase Chain Reaction technology (qPCR) can be affordably adapted to produce high-throughput results through a single-cell approach. In addition to comparative expression profiles between groups, single-cell approaches allow us to evaluate and propose new dependency relationships among markers. However, this alternative has not been explored before for large-scale qPCR-based experiments. RESULTS: Herein, we present deltaXpress (ΔXpress), a web app for analyzing data from single-cell qPCR experiments using a combination of HTML and R programming languages in a friendly environment. This application uses cycle threshold (Ct) values and categorical information for each sample as input, allowing the best pair of housekeeping genes to be chosen to normalize the expression of target genes. ΔXpress emulates a bulk analysis by observing differentially expressed genes, but in addition, it allows the discovery of pairwise genes differentially correlated when comparing two experimental conditions. Researchers can download normalized data or use subsequent modules to map differentially correlated genes, perform conventional comparisons between experimental groups, obtain additional information about their genes (gene glossary), and generate ready-to-publication images (600 dots per inch). CONCLUSIONS: ΔXpress web app is freely available to non-commercial users at https://alexismurillo.shinyapps.io/dXpress/ and can be used for different experiments in all technologies involving qPCR with at least one housekeeping region.
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Perfilação da Expressão Gênica , Linguagens de Programação , Perfilação da Expressão Gênica/métodos , Genes EssenciaisRESUMO
The perinatal period and early infancy are considered critical periods for lung development. During this period, adversities such as environmental exposures, allergic sensitization, and asthma are believed to impact lung health in adulthood. Therefore, we hypothesized that concomitant exposure to allergic sensitization and urban-derived fine particulate matter (PM2.5) in the early postnatal period of mice would cause more profound alterations in lung alveolarization and growth and differently modulate lung inflammation and gene expression than either insult alone in adult life. BALB/c mice were sensitized with ovalbumin (OVA) and exposed to PM2.5 from the fifth day of life. Then, we assessed lung responsiveness, inflammation in BALF, lung tissue, and alveolarization by stereology. In addition, we performed a transcriptomic analysis of lung tissue on the 40th day of life. Our results showed that young adult mice submitted to allergic sensitization and exposure to ambient PM2.5 since early life presented decreased lung growth with impaired alveolarization, a mixed neutrophilic-eosinophilic pattern of lung inflammation, increased airway responsiveness, and increased expression of genes linked to neutrophil recruitment when compared to animals that were OVA-sensitized or PM2.5 exposed only. Both, early life allergic sensitization and PM2.5 exposure, induced inflammation and impaired lung growth, but concomitant exposure was associated with worsened inflammation parameters and caused alveolar enlargement. Our experimental data provide pathological support for the hypothesis that allergic or environmental insults in early life have permanent adverse consequences for lung growth. In addition, combined insults were associated with the development of a COPD-like phenotype in young adult mice. Together with our data, current evidence points to the urgent need for healthier environments with fewer childhood disadvantage factors during the critical windows of lung development and growth.
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Poluentes Atmosféricos , Poluição do Ar , Pneumonia , Doença Pulmonar Obstrutiva Crônica , Poluentes Atmosféricos/análise , Poluição do Ar/efeitos adversos , Animais , Líquido da Lavagem Broncoalveolar , Inflamação/induzido quimicamente , Pulmão , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina , Material Particulado/análise , Fenótipo , Pneumonia/induzido quimicamenteRESUMO
Radiation induces the generation of platelet-activating factor receptor (PAF-R) ligands, including PAF and oxidized phospholipids. Alternatively, PAF is also synthesized by the biosynthetic enzymes lysophosphatidylcholine acyltransferases (LPCATs) which are expressed by tumor cells including melanoma. The activation of PAF-R by PAF and oxidized lipids triggers a survival response protecting tumor cells from radiation-induced cell death, suggesting the involvement of the PAF/PAF-R axis in radioresistance. Here, we investigated the role of LPCATs in the melanoma cell radiotherapy response. LPCAT is a family of four enzymes, LPCAT1-4, and modular nucleic acid nanoparticles (NANPs) allowed for the simultaneous silencing of all four LPCATs. We found that the in vitro simultaneous silencing of all four LPCAT transcripts by NANPs enhanced the therapeutic effects of radiation in melanoma cells by increasing cell death, reducing long-term cell survival, and activating apoptosis. Thus, we propose that NANPs are an effective strategy for improving radiotherapy efficacy in melanomas.
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1-Acilglicerofosfocolina O-Aciltransferase , Inativação Gênica , Melanoma , Nanopartículas , Proteínas de Neoplasias , Ácidos Nucleicos , 1-Acilglicerofosfocolina O-Aciltransferase/antagonistas & inibidores , 1-Acilglicerofosfocolina O-Aciltransferase/biossíntese , Linhagem Celular Tumoral , Humanos , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Nanopartículas/química , Nanopartículas/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/biossíntese , Ácidos Nucleicos/química , Ácidos Nucleicos/farmacologiaRESUMO
BACKGROUND: Melanoma is the most lethal type of skin cancer. Since chemoresistance is a significant barrier, identification of regulators affecting chemosensitivity is necessary in order to create new forms of intervention. Prohibitin 1 (PHB1) can act as anti-apoptotic or tumor suppressor molecule, depending on its subcellular localization. Our recent data shown that accumulation of PHB1 protects melanoma cells from chemotherapy-induced cell death. Lacking of post-transcriptional regulation of PHB1 could explain this accumulation. Interestingly, most of melanoma patients have down-regulation of microRNA-195. Here, we investigate the role of miR-195, its impact on PHB1 expression, and on chemosensitivity in melanoma cells. METHODS: TCGA-RNAseq data obtained from 341 melanoma patient samples as well as a panel of melanoma cell lines were used in an expression correlation analysis between PHB1 and predicted miRNAs. miR-195 impact on PHB1 mRNA and protein levels and relevance of this regulation were investigated in UACC-62 and SK-MEL-5 melanoma lines by RT-qPCR and western blot, luciferase reporter and genetic rescue experiments. Cell proliferation, cell-cycle analysis and caspase 3/7 assay were performed to investigate the potential action of miR-195 as chemosensitizer in melanoma cells treated with cisplatin and temozolomide. RESULTS: Analysis of the TCGA-RNAseq revealed a significant negative correlation (Pearson) between miR-195 and PHB1 expression. Moreover, RT-qPCR data showed that miR-195 is down-regulated while PHB1 is up-regulated in a collection of melanoma cells. We demonstrated that miR-195 regulates PHB1 directly by RT-qPCR and western blot in melanoma cells and luciferase assays. To establish PHB1 as a relevant target of miR-195, we conducted rescue experiments in which we showed that PHB1 transgenic expression could antagonize the suppressive effect miR-195 on the proliferation of melanoma cells. Finally, transfection experiments combined with drug treatments performed in the UACC-62 and SK-MEL-5 melanoma cells corroborated miR-195 as potential anti-proliferative agent, with potential impact in sensitization of melanoma cell death. CONCLUSIONS: This study support the role of miR-195 as anti-proliferative miRNA via targeting of PHB1 in melanoma cells.
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Melanoma/tratamento farmacológico , Melanoma/genética , MicroRNAs/genética , Proteínas Repressoras/genética , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Cisplatino/administração & dosagem , Dacarbazina/administração & dosagem , Dacarbazina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/administração & dosagem , Masculino , Melanoma/patologia , Proibitinas , Sulfonamidas/administração & dosagem , Temozolomida , Transfecção , VemurafenibRESUMO
The 9p trisomy syndrome is a rare condition, clinically characterized by a wide range of dysmorphic features, intellectual disability, and, in most patients, by short stature. Recombinant human growth hormone (rhGH) therapy is still controversial in syndromic disorders, the reason for which it is not currently indicated. Here we report a 7-year-old boy with 9p trisomy syndrome and marked short stature. Results of routine laboratory assessments were normal. IGF1 and IGFBP3 levels were both in the normal range (-1.6 and -0.7 SDS, respectively). GH peak in response to oral clonidine stimulation test was 3.5 µg/L, which is considered a normal response. Chromosomal analysis revealed the karyotype 47,XY, + del(9)(pter-q11:) dn. SNP array data indicated absence of mosaicism [arr 9p24.3-p13.1 (203,861-38,787,480) x3]. By the age of 8.3 years, the patient had persistent short stature (-2.9 SDS) with normal growth velocity (4.9 cm/y; -0.7 SDS), not showing spontaneous catch-up. After 5.6 years of rhGH therapy (50 µg/kg/d), height SDS improved from -2.9 to -1.0. This result suggests that rhGH therapy could be considered for patients with 9p trisomy syndrome who present with short stature. The degree of intellectual disability and the potential for social inclusion should be taken into account when recommending this treatment. Additional studies are needed to establish the benefits of height gain in these patients.
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Transtornos Cromossômicos/tratamento farmacológico , Transtornos Cromossômicos/genética , Hormônio do Crescimento Humano/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Trissomia , Criança , Transtornos Cromossômicos/diagnóstico , Cromossomos Humanos Par 9 , Nanismo/tratamento farmacológico , Nanismo/genética , Fácies , Gráficos de Crescimento , Humanos , Cariotipagem , Masculino , Fenótipo , Polimorfismo de Nucleotídeo Único , Resultado do Tratamento , Trissomia/diagnósticoRESUMO
Background: Capsular contracture is a common complication affecting about 80% of patients who receive radiotherapy after breast reconstruction with silicone prostheses. This study examines the use of adipocyte stem cells (ASCs) to treat capsular contracture. Methods: Thirty rats were operated on to implant a minisilicone prosthesis in the dorsal region. The rats were divided into three groups: control (saline solution injection), radiotherapy (RDT), and RDT + ASC. After 3 months, the capsules were collected and submitted to histological analysis for inflammatory cell presence, vascular density, and collagen fibers, and gene expression of Tnf, Il1rap, Il10, Cd68, Mmp3, and Mmp9 by qPCR. Results: In macroscopic analysis, the RTGO score showed a two-point reduction in RDT + ASC compared with the RDT (P = 0.003). In histological analysis, ASC exhibited less than 50% of inflammatory cells compared with RDT (P = 0.004), which was similar to control. This study demonstrated that Il1rap gene expression was identical in both RDT and RTD + ASC. Compared with control, treatment with ASC reduced Il1rap expression by 30%. Cd68 and Mmp3 expression levels were similar in both the control and RTD + ASC. Conclusion: This study suggests that ASC treatment decreases silicone prosthesis capsule inflammation.
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BACKGROUND AND AIMS: Peroxisome proliferator-activated receptor α is a nuclear receptor involved in the regulation of several biochemical pathways. Polymorphisms within its gene have been associated with several metabolic traits. We aimed to investigate the association of L162V and Intron 7G>C polymorphisms with serum level markers and common morbidities affecting an older adult/elderly cohort from Cuiaba City, Mato Grosso State, Brazil, as well as to compare the results with a previously studied population from São Paulo City, Brazil. METHODS AND RESULTS: The studied population consisted of 570 subjects from Cuiaba City, Brazil, who were subjected to clinical interviews and blood collection for laboratory examinations and DNA extraction. Dyslipidemia was defined when participants were taking oral hypolipemiants or those with total cholesterol above 200mg/dL, HDL-c below 40 mg/dL, LDL-c above 130 mg/dL and TG above 150 mg/dL. Restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) was used for polymorphism genotyping. Individual polymorphism and haplotype data were available for analyses. In the studied sample, allele frequencies were 0.052 and 0.292 for 162V and Intron 7C, respectively. In brief, 162V allele was associated with dyslipidemia (p=0.025), and after correction for alcohol consumption and waist-to-rip ratio, a tendency of association could still be observed (p=0.050). In addition, Intron 7C allele was associated with dyslipidemia even after correction for the same variables (p=0.029). When compared to our previous study from São Paulo, we found some divergences regarding these results, which may be explained by differences between the two populations. Haplotype association analyses revealed an association between L/C haplotype and dyslipidemia (p=0.021) and between V/C haplotype and lower LDL-c levels when compared to L/G haplotype (p=0.044). CONCLUSION: These results may help to clarify the role of PPARα gene in lipid and lipoprotein metabolism and the evaluation of its polymorphisms and haplotypes as being characterized as genetic risk factors for metabolic disturbances.
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Dislipidemias/epidemiologia , Dislipidemias/genética , PPAR alfa/genética , Polimorfismo de Nucleotídeo Único , Idoso , Idoso de 80 Anos ou mais , Brasil/epidemiologia , LDL-Colesterol/metabolismo , Feminino , Frequência do Gene , Genótipo , Humanos , Desequilíbrio de Ligação , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
Penile cancer (PeC) carcinogenesis is not fully understood, and no biomarkers are reported in clinical practice. We aimed to investigate molecular signatures based on miRNA and mRNA and perform an integrative analysis to identify molecular drivers and pathways for PeC development. Affymetrix miRNA microarray was used to identify differentially expressed miRNAs (DEmiRs) comparing 11 tumoral tissues (TT) paired with non-neoplastic tissues (NNT) with further validation in an independent cohort (n = 13). We also investigated the mRNA expression of 83 genes in the total sample. Experimentally validated targets of DEmiRs, miRNA-mRNA networks, and enriched pathways were evaluated in silico. Eight out of 69 DEmiRs identified by microarray analysis were validated by qRT-PCR (miR-145-5p, miR-432-5p, miR-487b-3p, miR-30a-5p, miR-200a-5p, miR-224-5p, miR-31-3p and miR-31-5p). Furthermore, 37 differentially expressed genes (DEGs) were identified when comparing TT and NNT. We identified four downregulated DEmiRs (miR-30a-5p, miR-432-5p, miR-487b-3p, and miR-145-5p) and six upregulated DEGs (IL1A, MCM2, MMP1, MMP12, SFN and VEGFA) as potential biomarkers in PeC by their capacity of discriminating TT and NNT with accuracy. The integration analysis showed eight dysregulated miRNA-mRNA pairs in penile carcinogenesis. Taken together, our findings contribute to a better understanding of the regulatory roles of miRNAs and altered transcripts levels in penile carcinogenesis.
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Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear transcription factor strictly involved in lipid and lipoprotein metabolisms. Thus, PPARalpha gene polymorphisms have been investigated as cardiovascular risk factors. We aimed to investigate associations of L162V and intron 7G>C polymorphisms with common morbidities affecting a Brazilian elderly cohort as well as with lipid and protein serum levels. Genotyping was performed by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP), and allele frequencies were determined. In addition, we performed the linkage disequilibrium analysis. Descriptive statistics, logistic regression analysis, and Student's t-test were used. Rare alleles for L162V and intron 7 G>C polymorphisms showed frequencies of 0.047 and 0.199, respectively. Our data showed that these polymorphisms were in linkage disequilibrium (p=0.0002). Intron 7 G>C polymorphism presented a tendency of association with neoplasia (p=0.053), and C allele was associated with higher HDL (p=0.010), lower triglycerides (p=0.001), and VLDL levels (p=0.003) compared to G allele. These data might suggest a protective role of intron 7 G>C polymorphism in the development of cardiovascular diseases and will help to clarify the importance of PPARalpha polymorphisms as key modulators of lipid metabolism in Brazilian population.
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Predisposição Genética para Doença , Hiperlipidemias/genética , Lipídeos/sangue , PPAR alfa/genética , Polimorfismo de Nucleotídeo Único/genética , Idoso , Idoso de 80 Anos ou mais , Brasil/epidemiologia , DNA/sangue , Feminino , Frequência do Gene , Genótipo , Humanos , Hiperlipidemias/sangue , Hiperlipidemias/epidemiologia , Desequilíbrio de Ligação , Lipoproteínas/sangue , Masculino , Polimorfismo de Fragmento de Restrição , Triglicerídeos/sangueRESUMO
Extracellular vesicles (EVs) are emerging as key players in intercellular communication. EVs can transfer biological macromolecules to recipient cells, modulating various physiological and pathological processes. It has been shown that tumor cells secrete large amounts of EVs that can be taken up by malignant and stromal cells, dictating tumor progression. In this study, we investigated whether EVs secreted by melanoma cells in response to chemotherapy modulate tumor response to alkylating drugs. Our findings showed that human and murine melanoma cells secrete more EVs after treatment with temozolomide and cisplatin. We observed that EVs shed by melanoma cells after temozolomide treatment modify macrophage phenotype by skewing macrophage activation towards the M2 phenotype through upregulation of M2-marker genes. Moreover, these EVs were able to favor melanoma re-growth in vivo, which was accompanied by an increase in Arginase 1 and IL10 gene expression levels by stromal cells and an increase in genes related to DNA repair, cell survival and stemness in tumor cells. Taken together, this study suggests that EVs shed by tumor cells in response to chemotherapy promote tumor repopulation and treatment failure through cellular reprogramming in melanoma cells.
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Antineoplásicos Alquilantes/farmacologia , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/fisiologia , Melanoma/tratamento farmacológico , Melanoma/fisiopatologia , Temozolomida/farmacologia , Animais , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/efeitos dos fármacos , Micropartículas Derivadas de Células/patologia , Micropartículas Derivadas de Células/fisiologia , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/fisiologia , Cisplatino/farmacologia , Progressão da Doença , Vesículas Extracelulares/patologia , Expressão Gênica/efeitos dos fármacos , Humanos , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/fisiologia , Melanoma/patologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Melanoma Experimental/fisiopatologia , Camundongos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologiaRESUMO
BACKGROUND: Multi-ethnicity of Brazilian population displays high levels of genomic diversity. Polymorphism may detect people at higher risk of developing cancer, distinctive response to treatment, and prognosis. Cyclooxygenase-2 (COX-2) is induced in response to growth factors and cytokines, and is expressed in inflammatory diseases, precancerous lesions and colorectal cancer (CRC). The aim of this study was to evaluate the influence of COX-2 -1195A > G and 8473T > C polymorphisms as a risk factor of developing CRC. METHODS: We evaluated COX-2 Single Nucleotide Polymorphism (SNP) of 230 CRC patients and 196 healthy controls by Real-Time Polymerase Chain Reaction. RESULTS: Populations were in Hardy-Weinberg equilibrium (HWE), except for control group of 8473T > C SNP. The frequencies were similar in both groups for genotypes and haplotypes. There was no association between studied polymorphisms and risk of CRC. CONCLUSIONS: The gene polymorphisms studied do not participate in the genetic susceptibility to CRC in a Brazilian population.
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The loss of cholinergic transmission is considered to be an important cause of Alzheimer's disease (AD). Treatment with acetyl cholinesterase inhibitors (ChEIs) shows benefits; however, great heterogeneity has been observed in patient responses. We evaluated apolipoprotein E (APOE) and α7 nicotinic receptor (CHRNA7) single-nucleotide polymorphisms (SNPs) and associated these SNPs with pharmacological responses to ChEIs in a Brazilian population with AD. We studied 177 outpatients using ChEIs, and they were classified as responders and nonresponders according to variation in Mini-Mental State Examination (MMSE) status. The analysis of APOE genotypes showed that patients with the ε4 allele had a worse response than those without the ε4 allele. We observed an association between the CHRNA7 T allele and a better response to treatment with ChEIs in patients with mild AD (MMSE ≥ 20). The SNP rs6494223 of CHRNA7 as well as APOEε4 could be useful for understanding the response to ChEI treatment in patients with AD.
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Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Apolipoproteína E4/genética , Inibidores da Colinesterase/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/genética , Idoso , Idoso de 80 Anos ou mais , Brasil , Feminino , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND AND AIMS: The characterization of candidate gene polymorphisms in elderly populations is an important tool for the identification of risk factors for age-related diseases and conditions. We aimed to genotype the APOE polymorphisms (rs429358 and rs7412), rs61886492 (1561C>T) and rs202720 of GCPII gene and rs3918242 (-1562C>T) of MMP9 gene in an older-adult/elderly cohort from Cuiabá city, Mato Grosso Brazil as well as to characterize risk factors for morbidities and conditions affecting this cohort. METHODS: The studied population consisted of 570 subjects from Cuiabá city, Brazil, who were subjected to clinical interviews and blood collection for laboratory examinations and DNA extraction. Restriction Fragment Length Polymorphism Polymerase Chain Reaction (RFLP-PCR), sequence-specific primer PCR (SSP-PCR) and TaqMan® allelic discrimination assay were used for genotyping. RESULTS: The frequencies of APOE ε2 and ε4 were 6.6% and 14.8%, respectively, and the frequencies of GCPII rs61886492 T allele, GCPII rs202720 C allele and MMP9 rs3918242 T allele were, respectively, 3.0%, 26.6% and 10.1%. Significant associations between APOE ε2 allele with lower total cholesterol and LDL-cholesterol were found. In addition, MMP9 rs3918242 T allele was associated with higher LDL-cholesterol levels, suggesting a link between lipid metabolism alteration and cardiovascular disease. CONCLUSIONS: The present findings contributed to characterize risk factors specific for the studied population and to better understand the molecular physiopathology of common morbidities and conditions affecting older-adult/elderly people.
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Apolipoproteínas E/genética , Carboxipeptidases/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Lipídeos/sangue , Metaloproteinase 9 da Matriz/genética , Polimorfismo Genético , Fatores Etários , Idoso , Alelos , Brasil , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Alzheimer's disease (AD) is the most common form of dementia in elderly. Chaperones may have a crucial role in AD due to their involvement in protein quality control, folding, and degradation. In this study, we investigated the mRNA and promoter DNA methylation levels of two chaperones, HSPA8 and HSPA9, in postmortem brain tissue (entorhinal and auditory cortices and hippocampus) from healthy elderly and AD subjects as well as in peripheral blood of healthy elderly and AD patients. mRNA quantification was performed by qRT-PCR and DNA methylation by mass spectrometry. In the peripheral blood, we did not observe a significant difference in HSPA8 and HSPA9 expression between elderly controls and AD. A significant downregulation of HSPA8 and HSPA9 was observed in AD across the three brain regions compared to the controls, suggesting their participation in AD pathogenesis. However, no important DNA methylation differences were observed, suggesting that other mechanism may be involved in controlling these genes expression.
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Doença de Alzheimer , Encéfalo/patologia , Metilação de DNA/fisiologia , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas Mitocondriais/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Análise de Variância , Apolipoproteína E4/genética , Encéfalo/metabolismo , Distribuição de Qui-Quadrado , Feminino , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteínas Mitocondriais/metabolismo , RNA Mensageiro/genéticaRESUMO
Alzheimer's disease (AD) is a highly prevalent type of dementia in the elderly population. AD is a complex neurodegenerative disorder. Thus, epigenetic mechanisms that regulate gene expression might have an important role in AD. CNP (2',3'-Cyclic Nucleotide 3' Phosphodiesterase) gene encodes a protein used as an index of myelin alterations. DPYSL2 (Dihydropyrimidinase-like 2) is described as acting in structural and regulatory processes in the central nervous system, such as neural differentiation, neurotransmitter release, and stabilization of microtubules. In this study, we evaluated gene expression and epigenetic regulation of CNP and DPYSL2 genes in three postmortem brain regions (entorhinal and auditory cortices and hippocampus) of AD patients and healthy elderly controls. mRNA quantification was performed using qRT-PCR, and promoter DNA methylation patterns were determined by mass spectrometry using the Sequenom EpiTYPER platform. We observed CNP mRNA downregulation in entorhinal and auditory cortex in relation to the same regions of the control group. CNP alterations in the brain might suggest impairment in myelination leading to a synaptic and cognition loss. No AD-associated differences in CNP and DPYSL2 promoter DNA methylation were observed, suggesting that other mechanisms may be involved in mediating the observed CNP gene expression.
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2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/genética , Doença de Alzheimer/genética , Metilação de DNA/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas/genética , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Apolipoproteínas E/genética , Sequência de Bases , Encéfalo/fisiologia , Feminino , Expressão Gênica/fisiologia , Frequência do Gene , Genótipo , Humanos , Masculino , Dados de Sequência Molecular , RNA Mensageiro/metabolismoRESUMO
Alzheimer's Disease (AD) is a neurodegenerative disorder and the most common cause of dementia among the elderly. Efforts have been made to understand the genetic and epigenetic mechanisms involved in the development of this disease. As SORL1 (sortilin-related receptor) and SIRT1 (sirtuin 1) genes have been linked to AD pathogenesis, we aimed to investigate their mRNA expression and promoter DNA methylation in post mortem brain tissues (entorhinal and auditory cortices and hippocampus) from healthy elderly subjects and AD patients. We also evaluated these levels in peripheral blood leukocytes from young, healthy elderly and AD patients, investigating whether there was an effect of age on these profiles. The comparative CT method by Real Time PCR and MALDI-TOF mass spectrometry were used to analyze gene expression and DNA methylation, respectively. SORL1 gene was differently expressed in the peripheral blood leukocytes and might act as a marker of aging in this tissue. Furthermore, we found that SORL1 promoter DNA methylation might act as one of the mechanisms responsible for the differences in expression observed between blood and brain for both healthy elderly and AD patients groups. The impact of these studied genes on AD pathogenesis remains to be better clarified.
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Envelhecimento/genética , Doença de Alzheimer/genética , Metilação de DNA , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas de Membrana Transportadoras/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Sirtuína 1/genética , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/fisiopatologia , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
INTRODUCTION: APOA1/C3/A4/A5 gene cluster is closely involved in lipid metabolism, and its polymorphisms have been associated with coronary heart disease and lipid plasma levels. Here, we aimed to investigate associations of APOC3 (3238C>G, -482C>T, 1100C>T) and APOA4 (Gln360His, Thr347Ser) polymorphisms in 382 individuals from a cohort of a Longitudinal Brazilian Elderly Study with major age-related morbidities and with lipid and protein serum levels. MATERIALS AND METHODS: The whole sample was genotyped by polymerase chain reaction-restriction fragment length polymorphism. Descriptive statistics, logistic regression analysis, Student t test, deviation from Hardy-Weinberg, Bonferroni correction for multiple testing, and haplotype analyses were performed. RESULTS: Although APOC3 1100T allele carriers presented lower triglyceride and very low density lipoprotein levels than non-T carriers, these associations disappeared after Bonferroni correction (P > 0.05). Moreover, APOA4 360His allele was associated with depression (P = 0.03), increased triglyceride (P = 0.035) and very low density lipoprotein (P = 0.035) levels, and reduced HDL levels (P = 0.0005). Haplotype analyses found an association between His/C/C haplotype (Gln360His/-482C>T/1100C>T) with depression, but this result was due to Gln360His polymorphism. CONCLUSIONS: Our data suggest that 360His allele might be a risk factor for depression and unfavorable lipid profile and depression for elderly people in the Brazilian population.
Assuntos
Apolipoproteínas A/genética , Lipídeos/sangue , Polimorfismo Genético , Idoso , Alelos , Apolipoproteínas A/sangue , Brasil , Estudos Transversais , Depressão/sangue , Depressão/genética , Feminino , Genótipo , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição , Fatores de Risco , Triglicerídeos/genética , Triglicerídeos/metabolismoRESUMO
Genetic variations in the APOA1/C3/A4/A5 gene cluster have been studied and proposed to be the leading key for susceptibility to cardiovascular diseases and age-associated disorders. We aimed to investigate the associations of rs12721026 (APOA1) and rs1729408 (APOA5) polymorphisms and their haplotypes with some age-related diseases, as well as with lipids and proteins serum levels in a cohort from a Brazilian Elderly Longitudinal Study (EPIDOSO). Genotyping was performed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Statistical analyses were carried out using logistic regression analysis, Student's t-test, and linkage disequilibrium (LD) analysis. Polymorphic allele frequencies were 0.095 and 0.449 for rs12721026 and rs1729408, respectively. The C-allele of rs1729408 was associated with higher high-density lipoprotein (HDL) (P = 0.022) and glycated hemoglobin levels (P = 0.020). We also showed that rs12721026 and rs1729408 were in LD. The GC haplotype, which is composed of the G-allele of rs12721026 and the C-allele of rs1729408, was significantly associated with obesity (P = 0.028), with higher glycated hemoglobin (P = 0.006), and fasting glucose (P = 0.0003) compared to the TT haplotype, which includes the wild-type alleles of both polymorphisms. Moreover, we found an association between the TC haplotype and higher HDL levels (P = 0.0039). This is the first time that haplotypes involving these polymorphisms were evaluated. Our results showed that these polymorphisms were involved in the development of obesity and in alterations of lipids and proteins serum levels in a Brazilian population. The present findings might also clarify the role of these polymorphisms and their haplotypes in lipids and proteins metabolism.
Assuntos
Apolipoproteína A-I/genética , Apolipoproteínas A/genética , Variação Genética , Haplótipos , Obesidade/genética , Apolipoproteína A-V , Brasil , Estudos de Coortes , Frequência do Gene , Genótipo , Humanos , Desequilíbrio de Ligação , Lipídeos/sangue , Lipídeos/genética , Obesidade/epidemiologia , Fatores de RiscoRESUMO
Several polymorphisms in apolipoprotein A1 (APOA1) gene have been associated with metabolic diseases. Increased transcription efficiency was observed in -75A allele carriers compared to -75G allele homozygotes. +83C allele was associated with higher body mass index and waist-to-hip ratio in type II diabetes subjects. -75G/A and +83C/T polymorphisms were analyzed by RFLP-PCR in 334 individuals from a Brazilian elderly cohort. APOA1 polymorphisms were associated with age-related morbidities, as well as with triglycerides, total cholesterol, HDL, VLDL, LDL, creatinine, urea, albumin, glycated hemoglobin and fasting glucose serum levels. Allele frequencies were 0.102 and 0.21, respectively, for -75A and +83T. -75G allele showed significant association with hypertension (P = 0.001). An association between +83C allele and obesity was observed (P = 0.040) and this allele also showed an association with hypertension in the presence of cardiovascular disease (P = 0.047). Moreover, +83T allele was associated with lower glycated hemoglobin values (P = 0.026). To our knowledge, there is no data associating this polymorphism with glycated hemoglobin. Furthermore, individuals carrying AT haplotype have lower risk for developing hypertension (P = 0.0002), while GT haplotype carriers present decreased risk to develop obesity comparing to GC haplotype (P = 0.025). APOA1 polymorphisms analysis may be a useful tool to identify risk factors for subjects and families and clarify the physiopathological role of these polymorphisms in age-related diseases, such as hypertension and obesity.