RESUMO
BACKGROUND: Age-related macular degeneration is a progressive eye disease affecting the macula and causing acute visual loss particularly in elder people. The aim of the study was an attempt to discern an influence of expression levels and functional genetic polymorphisms of selected genes related to the extracellular matrix turnover or neovascularization on age-related macular degeneration occurrence and progression. METHODS: We conducted a case-control study of 200 polish patients with recognized age-related macular degeneration (dry and wet) and compared the results with those obtained from matched 100 healthy control subjects. TaqMan Genotyping Assays were employed to examine the following single nucleotide polymorphisms: matrix metalloproteinase (MMP)-2 -735C/T, MMP-7 -181A/G, MMP-9 -1702T/A, and -1562C/T; tissue inhibitors of metalloproteinase (TIMP)-2 -418G/C; vascular endothelial growth factor (VEGF) +405 G/C and +936 C/T, VEGFR-2 +1719 T/A and -271 G/A. Real-time polymerase chain reaction was assessed to determine the mRNA quantity. Serum levels of proteins were measured using enzyme-linked immunosorbent assay. RESULTS: The single nucleotide polymorphism genotyping showed that TT genotype for MMP-9 -1702T/A and CC genotype for VEGF +936C/T increase markedly the risk of age-related macular degeneration but do not influence on its progression. Additionally, the possible protective effect of CC genetic variant in MMP-9 -1562C/T polymorphism against progression of age-related macular degeneration was observed. We also found significant differences in systemic expression levels of MMP-2, -7, -9, TIMP-2, vascular endothelial growth factor, VEGFR-2, and pigment epithelium-derived factor between studied group. The research demonstrated evident differences in serum levels of MMP-2, -7, -9, TIMP-2, vascular endothelial growth factor, and pigment epithelium-derived factor between wet and dry age-related macular degeneration patients. CONCLUSIONS: We can conclude that disturbances in angiogenic homeostasis and processes of extracellular matrix turnover occurring in age-related macular degeneration-affected ocular tissues may be reflected in changes in systemic expression levels of the investigated genes.
Assuntos
Matriz Extracelular/enzimologia , Degeneração Macular/genética , Metaloproteinases da Matriz/genética , Polimorfismo de Nucleotídeo Único , Neovascularização Retiniana/genética , Inibidor Tecidual de Metaloproteinase-2/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação Enzimológica da Expressão Gênica/fisiologia , Técnicas de Genotipagem , Humanos , Degeneração Macular/diagnóstico , Degeneração Macular/enzimologia , Masculino , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 7 da Matriz/sangue , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 9 da Matriz/sangue , Metaloproteinase 9 da Matriz/genética , Metaloproteinases da Matriz/sangue , Pessoa de Meia-Idade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Neovascularização Retiniana/diagnóstico , Neovascularização Retiniana/enzimologia , Inibidor Tecidual de Metaloproteinase-2/sangue , Fator A de Crescimento do Endotélio Vascular/sangue , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/sangueRESUMO
BACKGROUND Congenital hemochromatosis is a disorder caused by mutations of genes involved in iron metabolism, leading to increased levels of iron concentration in tissues and serum. High concentrations of iron can lead to the development of AMD. The aim of this study was to analyze circulating miRNAs in the serum of congenital hemochromatosis patients with AMD and their correlation with the expression of genes involved in iron metabolism. MATERIAL AND METHODS Peripheral blood monolayer cells and serum were obtained from patients with congenital hemochromatosis, congenital hemochromatosis and AMD, AMD patients without congenital hemochromatosis, and healthy controls. Serum miRNAs expressions were analyzed by RT-PCR (qRT-PCR) using TaqMan MicroRNA probes, and proteins levels were measured by ELSA kits. Gene polymorphisms in TF and TFRC genes were determined using the TaqMan discrimination assay. RESULTS Statistical analysis of the miRNAs expressions selected for further study the miR-31, miR-133a, miR-141, miR-145, miR-149, and miR-182, which are involved in the posttranscriptional expression of iron-related genes: TF, TFRI, DMT1, FTL, and FPN1. It was discovered that the observed changes in the expressions of the miRNAs was correlated with the level of protein in the serum of the analyzed genes. There were no statistically significant differences in the distribution of genotype and allele frequencies in TF and TFRC genes between analyzed groups of patients. CONCLUSIONS The differences studied in the miRNA serum profile, in conjunction with the changes in the analyzed protein levels, may be useful in the early detection of congenital hemochromatosis in patients who may develop AMD disease.
Assuntos
Antígenos CD/genética , Proteínas de Transporte de Cátions/genética , Hemocromatose/genética , Receptores da Transferrina/genética , Idoso , Idoso de 80 Anos ou mais , Alelos , Antígenos CD/metabolismo , Estudos de Casos e Controles , Proteínas de Transporte de Cátions/metabolismo , Feminino , Perfilação da Expressão Gênica , Frequência do Gene/genética , Hemocromatose/metabolismo , Humanos , Ferro/sangue , Ferro/metabolismo , Degeneração Macular/genética , Masculino , MicroRNAs/análise , MicroRNAs/sangue , MicroRNAs/genética , Pessoa de Meia-Idade , Polimorfismo Genético/genética , Receptores da Transferrina/metabolismo , Transferrina/genéticaRESUMO
BACKGROUND: Age-related macular degeneration (AMD) is a major cause of blindness worldwide. Circulating microRNAs (miRNAs) in serum have emerged as novel candidate biomarkers for many diseases. The aim of the present study was to identify a serum microRNA (miRNA) expression profile specific for dry and wet forms of AMD. MATERIAL AND METHODS: Serum miRNA expression was first screened using TaqMan® Human MicroRNA Array A (Applied Biosystems). An extensive, self-validated, individual, quantitative RT-PCR (qRT-PCR) study was then performed on a cohort of 300 AMD patients (150 wet form and 150 dry form) and 200 controls. The Mann-Whitney U test and nonparametric Spearman's rank correlation coefficient were used for statistical analysis. RESULTS: miRNA expression analysis revealed increased expression of miR661 and miR3121 in serum of patients with dry AMD and miR4258, miR889, and Let7 in patients with wet form. Expression of analyzed miRNA was not observed or remained at low level in controls. CONCLUSIONS: Differences in miRNA serum profile exist between patients with wet and dry form of AMD, which indicates miRNAs as potential biomarkers of AMD. Further studies should be performed to confirm its significance in clinical practice.
Assuntos
Biomarcadores/sangue , Regulação da Expressão Gênica , Degeneração Macular/genética , MicroRNAs/sangue , Idoso , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Small interfering RNA (siRNA) gene therapy is a new molecular approach in the search for an efficient therapy for Alzheimer disease (AD), based on the principle of RNA interference. Reducing BACE activity can have great therapeutic potential for the treatment of AD. In this study, receptor-mediated delivery was used to deliver opioid peptide-conjugated BACE 1 to INR-32 human neuroblastoma cells. MATERIAL AND METHODS: An INR-32 human neuroblastoma cell line was stably transfected to express the APP cDNA coding fragment containing the predicted sites for cleavage by α, ß, or γ-secretase. This was then treated with BACE 1 siRNA to silence BACE gene expression. BACE gene transcription and translation was determined using BACE-1 siRNA cross-linked with opioid peptide, together with RT-PCR, Western blot analysis, and ELISA. RESULTS: Receptor-mediated delivery was used to introduce BACE1 siRNA to the APP - INR 32 human neuroblastoma cells. Decreased BACE mRNA and protein expression were observed after the cells were transfected with BACE1 siRNA. CONCLUSIONS: Delivery of BACE1 siRNA appears to specifically reduce the cleavage of APP by inhibiting BACE1 activity.
