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AIM: functional analysis of pcsk9 3'utr variants and mrna-mirna interactions were explored in patients with familial hypercholesterolemia (fh). MATERIALS & METHODS: PCSK9 3'UTR variants were identified by exon-targeted gene sequencing. Functional effects of 3'UTR variants and mRNA-miRNA interactions were analyzed using in silico and in vitro studies in HEK293FT and HepG2 cells. RESULTS: Twelve PCSK9 3'UTR variants were detected in 88 FH patients. c.*75C >T and c.*345C >T disrupted interactions with miR-6875, miR-4721 and miR-564. Transient transfection of the c.*345C >T decreased luciferase activity in HEK293FT cells. miR-4721 and miR-564 mimics reduced PCSK9 expression in HepG2 cells. CONCLUSION: PCSK9 c.*345C >T has a possible role as loss-of-function variant. miR-4721 and miR-564 downregulate PCSK9 and may be useful to improve lipid profile in FH patients.
Assuntos
MicroRNAs , Epigenômica , Hiperlipoproteinemia Tipo II , Pró-Proteína Convertase 9RESUMO
Aim: Functional analysis of PCSK9 3'UTR variants and mRNA-miRNA interactions were explored in patients with familial hypercholesterolemia (FH). Materials & methods:PCSK9 3'UTR variants were identified by exon-targeted gene sequencing. Functional effects of 3'UTR variants and mRNA-miRNA interactions were analyzed using in silico and in vitro studies in HEK293FT and HepG2 cells. Results: Twelve PCSK9 3'UTR variants were detected in 88 FH patients. c.*75C >T and c.*345C >T disrupted interactions with miR-6875, miR-4721 and miR-564. Transient transfection of the c.*345C >T decreased luciferase activity in HEK293FT cells. miR-4721 and miR-564 mimics reduced PCSK9 expression in HepG2 cells. Conclusion:PCSK9 c.*345C >T has a possible role as loss-of-function variant. miR-4721 and miR-564 downregulate PCSK9 and may be useful to improve lipid profile in FH patients.
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Hiperlipoproteinemia Tipo II/genética , MicroRNAs , Pró-Proteína Convertase 9/genética , RNA Mensageiro , Regiões 3' não Traduzidas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Variação Genética , Células HEK293 , Células Hep G2 , Humanos , Hiperlipoproteinemia Tipo II/metabolismo , Masculino , Pessoa de Meia-Idade , Pró-Proteína Convertase 9/metabolismo , Adulto JovemRESUMO
Aim: To explore the association of circulating miRNAs with adiposity, metabolic status and inflammatory biomarkers in patients with metabolic syndrome (MetS). Methods: Serum levels of 372 miRNAs were measured in patients with (n = 6) and without MetS (n = 6) by quantitative PCR array, and dysregulated miRNAs were validated in a larger cohort (MetS, n = 89; non-MetS, n = 144). Results: In the screening study, seven miRNAs were dysregulated in patients with MetS, and miR-421 remained increased in the validation study. miR-421 was associated with a high risk of MetS and insulin resistance and hypertension and correlated with glycated hemoglobin, triacylglycerols, high-sensitivity CRP, IL-6, resistin and adiponectin (p < 0.05). Conclusion: Circulating miR-421 is a potential biomarker for insulin resistance, metabolic dysregulation and inflammatory status in patients with MetS.
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Biomarcadores/sangue , Regulação da Expressão Gênica , Inflamação/patologia , Resistência à Insulina , Síndrome Metabólica/complicações , MicroRNAs/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína C-Reativa/análise , Feminino , Seguimentos , Humanos , Inflamação/sangue , Inflamação/etiologia , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Prognóstico , Resistina/sangue , Triglicerídeos/sangueRESUMO
ABSTRACT: To explore the association of circulating miRNAs with adiposity, metabolic status and inflammatory biomarkers in patients with metabolic syndrome (MetS). METHODS: Serum levels of 372 miRNAs were measured in patients with (n = 6) and without MetS (n = 6) by quantitative PCR array, and dysregulated miRNAs were validated in a larger cohort (MetS, n = 89; non-MetS, n = 144). RESULTS: In the screening study, seven miRNAs were dysregulated in patients with MetS, and miR-421 remained increased in the validation study. miR-421 was associated with a high risk of MetS and insulin resistance and hypertension and correlated with glycated hemoglobin, triacylglycerols, high-sensitivity CRP, IL-6, resistin and adiponectin (p < 0.05). CONCLUSION: Circulating miR-421 is a potential biomarker for insulin resistance, metabolic dysregulation and inflammatory status in patients with MetS.
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Síndrome Metabólica , Adiponectina , Adiposidade , Resistência à Insulina , MicroRNAs , InflamaçãoRESUMO
OBJECTIVES: We sought to assess the effects of a high loading dose of rosuvastatin (40 mg) on acute inflammatory response after coronary stenting. METHODS: Patients with stable coronary disease without statin use (≥7 days) and undergoing elective percutaneous coronary intervention (PCI) in a native coronary artery were randomized to receive a loading dose of rosuvastatin (n = 64) or not (n = 61). Blood samples were obtained before statin intake (time point A), 3 hours after medication (time point B), and 3 hours after PCI (time point C). The primary goal was the comparison in the variation of the serum inflammatory markers and their gene expression at the different time points between the two groups. RESULTS: Baseline clinical, angiographic, and procedural characteristics did not significantly differ between the groups, except for the more frequent use of postdilation in the control group (73.4% vs 90.2%; P=.02). Patients pretreated with statin showed a reduction in the serum levels of interleukin (IL)-1ß (Δ = -0.491 pg/mL; Pinteraction<.001), IL-6 (Δ = -0.209 pg/mL; Pinteraction<.001), and plasminogen activator inhibitor 1 (Δ = -1.573 pg/mL; Pinteraction<.001) as well as in their genetic expression, which was not observed in the control group. Regarding high-sensitivity c-reactive protein, there was no significant variation in its value from time point A to C in patients pretreated with statin (P=.58) while it significantly increased in the control group (P=.04). CONCLUSIONS: Among patients with stable coronary artery disease undergoing PCI with stents, pretreatment with high dose of rosuvastatin resulted in significant reduction in the serum levels of important inflammatory markers and their genetic expression.
Assuntos
Síndrome Coronariana Aguda , Doença da Artéria Coronariana , Inibidores de Hidroximetilglutaril-CoA Redutases , Inflamação , Intervenção Coronária Percutânea , Rosuvastatina Cálcica/administração & dosagem , Proteína C-Reativa/análise , Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/tratamento farmacológico , Doença da Artéria Coronariana/cirurgia , Humanos , Inflamação/sangue , Interleucina-1beta/sangue , Intervenção Coronária Percutânea/efeitos adversos , Inibidor 1 de Ativador de Plasminogênio/sangueRESUMO
Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc(2) 155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages.