RESUMO
Advanced glycation end products (AGEs) delay spontaneous apoptosis of monocytes and contribute to the development of inflammatory responses. However, the mechanism by which AGEs affect monocyte apoptosis is unclear. We studied the role of microRNA-214 (miR-214) and its target gene in AGE-induced monocytic apoptosis delay. Using microRNA (miRNA) microarray and stem-loop, quantitative RT-PCR assay, we studied genome-wide miRNA expression in THP-1 cells treated with or without AGEs. Significant upregulation of miR-214 was consistently observed in THP-1 and human monocytes treated with various AGEs, and AGE-induced monocytic miR-214 upregulation was likely through activation of receptor for AGEs. A striking increase in miR-214 was also detected in monocytes from patients with chronic renal failure. Luciferase reporter assay showed that miR-214 specifically binds to the phosphatase and tensin homolog (PTEN) mRNA 3'-untranslated region, implicating PTEN as a target gene of miR-214. PTEN expression is inversely correlated with miR-214 level in monocytes. Compared with normal monocytes, AGE-treated monocytes and monocytes from chronic renal failure patients exhibited lower PTEN levels and delayed apoptosis. Overexpression of pre-miR-214 led to impaired PTEN expression and delayed apoptosis of THP-1 cells, whereas knockdown of miR-214 level largely abolished AGE-induced cell survival. Our findings define a new role for miR-214-targeting PTEN in AGE-induced monocyte survival.
Assuntos
Apoptose/imunologia , Marcação de Genes , Produtos Finais de Glicação Avançada/fisiologia , MicroRNAs/biossíntese , Monócitos/imunologia , Monócitos/patologia , PTEN Fosfo-Hidrolase/metabolismo , Albumina Sérica/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Apoptose/genética , Morte Celular/genética , Morte Celular/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Regulação da Expressão Gênica/imunologia , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Produtos Finais de Glicação Avançada/genética , Humanos , Falência Renal Crônica/enzimologia , Falência Renal Crônica/metabolismo , Falência Renal Crônica/patologia , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Monócitos/metabolismo , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , Interferência de RNA/imunologia , Albumina Sérica/antagonistas & inibidores , Albumina Sérica/genética , Albumina Sérica Humana , Fatores de Tempo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genéticaRESUMO
A simple and rapid method for preparation of plasmid DNA from overnight incubation was introduced. It does not require any additional reagents; the incubation mixture containing recombinant plasmid DNA was just mixed with H2O and phenol/chloroform/isoamyl alcohol in certain ratio. After vortexing and spinning of the mixture, the supernatant could be directly loaded onto agarose gel and analyzed using electrophoresis. The whole preparation requires only 3-5 minutes. So to quickly screen recombinant clones, this method is better compared with traditional methods.
