RESUMO
BACKGROUND: Dicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs); depletion of Dicer was found to impair the migration of endothelial cells. METHODS: siRNA transfection, cell migration assay, real-time RT-PCR, chromatin immunoprecipitation, Western blotting, ELISA, caspase-3 activity assay, and annexin-V-FITC assay were utilized. RESULTS: Knockdown of Dicer impairs the migratory capacity of HEK293T cells and induces fibronectin-1. The upregulation of fibronectin-1 is dependent on Egr1. Fibronectin-1/Dicer double-knockdown cells showed a marked increase in apoptosis compared with fibronectin-1 single knockdown cells. CONCLUSIONS: Decreased Dicer expression induces fibronectin-1 expression via an Egr1-dependent manner. GENERAL SIGNIFICANCE: Our data suggest that upregulation of fibronectin-1 protects Dicer knockdown HEK293T cells against apoptosis.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Fibronectinas/genética , Técnicas de Silenciamento de Genes , Rim/fisiologia , Ribonuclease III/genética , Apoptose , Western Blotting , Linhagem Celular , Movimento Celular/fisiologia , Cromatina/genética , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Fibronectinas/fisiologia , Expressão Gênica , Humanos , Rim/embriologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
Hepatitis B virus (HBV) replicates in most tumor tissues of patients with HBV-associated hepatocellular carcinoma (HCC). In the present study, we have shown that the expression of HBV in the HCC cell lines, HepG2 and Huh7, down-regulated the expression of MHC class I-related molecule A (MICA), a ligand of the NKG2D receptor. Inhibition of HBV expression by small interference RNAs (siRNAs) in HepG2.2.15, a cell line that constitutively expresses HBV, induced up-regulation of MICA. The up-regulation of MICA increased the lysis of HepG2.2.15 cells by NK cells. Our results suggest that HBV compromises the innate immune system in HCC patients and that inhibition of HBV replication by siRNAs may enhance the antitumor immune response.
Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Antígenos de Histocompatibilidade Classe I/biossíntese , RNA Interferente Pequeno/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Células Matadoras Naturais/imunologiaRESUMO
5-Aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, exerts antitumor activity through induction of cell cycle arrest, apoptosis and DNA damage. In this study, we showed that MHC class I-related chain B (MICB), a ligand of the NKG2D receptor expressed by natural killer cells and activated CD8(+) T cells, was upregulated following 5-aza-dC treatment. The upregulation of MICB was accompanied by promoter DNA demethylation and DNA damage. Furthermore, the upregulation of MICB was partially prevented by pharmacological or genetic inhibition of ataxia telangiectasia mutated (ATM) kinase. Our results suggest that promoter DNA demethylation, in combination with DNA damage, contribute to the upregulation of MICB induced by 5-aza-dC.
