RESUMO
Death receptor 3 (DR3) belongs to the tumor necrosis factor (TNF) receptor superfamily, primarily found in lymphoid tissues. Reports have determined that DR3 may also be distributed in numerous types of tumors. Therefore, it is thought that DR3 may have an important role in the process of tumorigenesis. The aim of the present study was to observe the effect of silencing DR3 expression on hepatocarcinoma cell growth, apoptosis and invasion in order to elucidate the role of DR3 in tumor development. The hepatocarcinoma cell lines (HepG2, Huh7, SMMC7721 and Bel7402) and normal human liver cells (HL7702) were transfected with three stealth RNA interference (RNAi) sequences that target the DR3 gene. Reverse transcription quantitative polymerase chain reaction was used to detect the expression levels of DR3 in hepatocarcinoma cell lines and normal liver HL7702 cells. MTT assay and flow cytometry (FCM) were used to determine the rates of cell proliferation and apoptosis, respectively. Following silencing of the DR3 gene, western blot analysis was used to determine the protein expression of P53, Fas, Caspase8, nuclear factor kappalightchainenhancer of activated B cells (NFκB) and Caspase3. DR3 messenger RNA (mRNA) expression in hepatocarcinoma cell lines was significantly increased compared with that in the normal liver cell line. Three targeted DR3 gene small interfering RNAs significantly inhibited DR3 gene expression in Bel7402 cells at the nucleic acid level. AF02670.1_stealth_883 and cocktail demonstrated the most efficient inhibition of DR3 gene expression at 48 and 72 h following transfection, with mRNA inhibition rates of 89.46 and 92.75%, and 90.53 and 94.25% (P<0.01), respectively. Cell viability was significantly reduced by AF02670.1_stealth_883 and RNAi cocktail at 24, 48 and 72 h following transfection. The inhibition rates of cell proliferation were 50.76 and 61.76% (P<0.05) at 72 h following transfection. FCM revealed that AF02670.1_stealth_883 and RNAi cocktail also induced apoptosis in Bel7402 cells at 72 h following transfection. Reduction of NFκB and P53 levels was observed (P<0.05) in Bel7402 cells following DR3 silencing, whereas levels of Fas, Caspase3 and Caspase8 were markedly elevated (P<0.05). DR3 expression levels in hepatocellular carcinoma cells were significantly higher than those in normal cells. DR3 silencing effectively inhibited proliferation and invasion of hepatocellular carcinoma cells in vitro. However, silencing of the DR3 gene affect levels of apoptosis antigen3 ligand in cells, therefore indicating that it may be involved with other pathways that regulate apoptosis in HCCs. In conclusion, the results of the present study indicated that DR3 may be a promising therapeutic target molecule for further study of hepatocellular carcinoma gene therapy.
Assuntos
Membro 25 de Receptores de Fatores de Necrose Tumoral/metabolismo , Apoptose , Carcinoma Hepatocelular/patologia , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/patologia , NF-kappa B/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Membro 25 de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Membro 25 de Receptores de Fatores de Necrose Tumoral/genética , Proteína Supressora de Tumor p53/metabolismo , Receptor fas/metabolismoRESUMO
Currently, the most widely used photoresists in optical lithography are organic-based resists. The major limitations of such resists include the photon accumulation severely affects the quality of photolithography patterns and the size of the pattern is constrained by the diffraction limit. Phase-change lithography, which uses semiconductor-based resists such as chalcogenide Ge2Sb2Te5 films, was developed to overcome these limitations. Here, instead of chalcogenide, we propose a metallic resist composed of Mg58Cu29Y13 alloy films, which exhibits a considerable difference in etching rate between amorphous and crystalline states. Furthermore, the heat distribution in Mg58Cu29Y13 thin film is better and can be more easily controlled than that in Ge2Sb2Te5 during exposure. We succeeded in fabricating both continuous and discrete patterns on Mg58Cu29Y13 thin films via laser irradiation and wet etching. Our results demonstrate that a metallic resist of Mg58Cu29Y13 is suitable for phase change lithography, and this type of resist has potential due to its outstanding characteristics.