RESUMO
Ornithine decarboxylase 1 (ODC1), a metabolic enzyme critically involved in the polyamine biosynthesis, is commonly upregulated in hepatocellular carcinoma (HCC). Despite its altered expression in human HCC tissues, the molecular mechanism by which ODC1 alters the course of HCC progression and functions in HCC cell survival is unknown. Here we identified that silencing of ODC1 expression with small interfering (si) RNA causes inhibition of HCC cell growth through blockade of cell cycle progression and induction of apoptosis. Next, to obtain insights into the molecular changes in response to ODC1 knockdown, global changes in gene expression were examined using RNA sequencing. It revealed that 119 genes show same directional regulation (76 up- and 43 down-regulated) in both Huh1 and Huh7 cells and were considered as a common ODC1 knockdown signature. Particularly, we found through a network analysis that KLF2, which is known to inhibit PPARγ expression and adipogenesis, was commonly up-regulated. Subsequent Western blotting affirmed that the downregulation of ODC1 was accompanied by a decrease in the levels of PPARγ as well as of PARP-1, cyclin E1 and pro-caspase 9 delaying cell cycle progression and accelerating apoptotic signaling. Following the down-regulation of PPARγ expression, ODC1 silencing resulted in a strong inhibition in the expression of important regulators of glucose transport and lipid biogenesis, and caused a marked decrease in lipid droplet accumulation. In addition, ODC1 silencing significantly inhibited the growth of human HCC xenografts in nude mice. These findings indicate that the function of ODC1 is correlated with HCC lipogenesis and suggest that targeting ODC1 could be an attractive option for molecular therapy of HCC.
Assuntos
Carcinoma Hepatocelular/genética , Proliferação de Células/genética , Metabolismo dos Lipídeos/genética , Neoplasias Hepáticas/genética , Ornitina Descarboxilase/genética , Interferência de RNA , Animais , Apoptose/genética , Western Blotting , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Caspase 9/genética , Caspase 9/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Ciclina E/genética , Ciclina E/metabolismo , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Ornitina Descarboxilase/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Terapêutica com RNAi/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Histone deacetylase 2 (HDAC2) is a chromatin modifier involved in epigenetic regulation of cell cycle, apoptosis, and differentiation that is upregulated commonly in human hepatocellular carcinoma (HCC). In this study, we show that specific targeting of this HDAC isoform is sufficient to inhibit HCC progression. siRNA-mediated silencing of HDAC inhibited HCC cell growth by blocking cell-cycle progression and inducing apoptosis. These effects were associated with deregulation of HDAC-regulated genes that control cell cycle, apoptosis, and lipid metabolism, specifically, by upregulation of p27 and acetylated p53 and by downregulation of CDK6 and BCL2. We found that HDAC2 silencing in HCC cells also strongly inhibited PPARγ signaling and other regulators of glycolysis (ChREBPα and GLUT4) and lipogenesis (SREBP1C and FAS), eliciting a marked decrease in fat accumulation. Notably, systemic delivery of HDAC2 siRNA encapsulated in lipid nanoparticles was sufficient to blunt the growth of human HCC in a murine xenograft model. Our findings offer preclinical proof-of-concept for HDAC2 blockade as a systemic therapy for liver cancer.
Assuntos
Carcinoma Hepatocelular/genética , Histona Desacetilase 2/genética , Neoplasias Hepáticas/genética , Isoformas de Proteínas/genética , Animais , Apoptose/genética , Carcinoma Hepatocelular/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 6 Dependente de Ciclina/genética , Progressão da Doença , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/genética , Glicólise/genética , Células Hep G2 , Humanos , Metabolismo dos Lipídeos/genética , Lipogênese/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos SCID , PPAR gama/genética , Antígeno Nuclear de Célula em Proliferação/genética , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Regulação para Cima/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Even with the recent awareness of enterovirus 71 (EV71) as a major public health issue, there are no preventive or therapeutic agents that are effective against EV71 infection. Although FLICE-like inhibitory protein (FLIP) has been identified as a factor that modulates virus pathogenesis, there are no reports regarding its effects on EV71 infection. The aim of the present study was to identify whether FLIP influences EV71 pathogenesis and to understand the underlying mechanisms. Virus replication was markedly reduced in MRC5 cells preincubated with anti-FLIP peptides, and infected cells were rescued from the cytopathic effects of the virus. The anti-FLIP peptides induced autophagy by disrupting intrinsic FLIP functions. The antiviral activity of these peptides was reduced when autophagy was inhibited by treatment with siRNA targeted to beclin-1. Thus, the present study provides evidence that anti-FLIP peptides induce autophagy by inactivating cFLIP, and that this is associated with antiviral effects against EV71.
Assuntos
Autofagia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Enterovirus Humano A/patogenicidade , Antivirais/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/antagonistas & inibidores , Linhagem Celular , Efeito Citopatogênico Viral , Enterovirus Humano A/fisiologia , Humanos , Peptídeos/metabolismo , Replicação ViralRESUMO
Antiviral therapeutics are currently unavailable for treatment of coxsackievirus B3, which can cause life-threatening myocarditis. A modified small interfering RNA (siRNA) containing 5'-triphosphate, 3p-siRNA, was shown to induce RNA interference and interferon activation. We aimed to develop a potent antiviral treatment using CVB3-specific 3p-siRNA and to understand its underlying mechanisms. Virus-specific 3p-siRNA was superior to both conventional virus-specific siRNA with an empty hydroxyl group at the 5' end (OH-siRNA) and nonspecific 3p-siRNA in decreasing viral replication and subsequent cytotoxicity. A single administration of 3p-siRNA dramatically attenuated virus-associated pathological symptoms in mice with no signs of toxicity, and their body weights eventually reached the normal range. Myocardial inflammation and fibrosis were rare, and virus production was greatly reduced. A nonspecific 3p-siRNA showed relatively less protective effect under identical conditions, and a virus-specific OH-siRNA showed no protective effects. We confirmed that virus-specific 3p-siRNA simultaneously activated target-specific gene silencing and type I interferon signaling. We provide a clear proof of concept that coxsackievirus B3-specific 3p-siRNA has 2 distinct modes of action, which significantly enhance antiviral activities with minimal organ damage. This is the first direct demonstration of improved antiviral effects with an immunostimulatory virus-specific siRNA in coxsackievirus myocarditis, and this method could be applied to many virus-related diseases.
