RESUMO
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.
Assuntos
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.
Assuntos
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.
Assuntos
Síndrome Metabólica , Adiponectina , Adiposidade , Resistência à Insulina , MicroRNAs , InflamaçãoRESUMO
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.