RESUMO
PURPOSE: After excimer laser surgery, epidermal growth factor (EGF) plays an important role in injured corneal epithelial cell on myofibroblastic cell formation in corneal stroma. The purpose of the study is to investigate the precise mechanism of EGF on corneal wound healing, particularly on epithelial proliferation and migration. METHODS: In this study we applied small interference RNA (siRNA) to knock down the expression of eukaryotic translation initiation factor 5A (eIF5A) in corneal epithelial cells. The relative mRNA and protein expression of matrix metallopeptidase 9 (MMP9) and proliferating cell nuclear antigen (PCNA) was determined via real-time PCR and western blot analysis. The proliferative potential of EGF was evaluated via a proliferation assay using the measurement of (3)H-thymidine incorporation ((3)H-TdR). HCEpiC apoptosis was subjected to flow cytometric analysis. RESULTS: The results showed eIF5A expression was enhanced and there was a statistically significant increase in EGF treatment compared to the control group. Real-time PCR, western blot analysis, and the proliferation assay demonstrated significantly lower MMP9 and PCNA expression and proliferation cell counts in eIF5A siRNA-treated groups versus significantly higher levels in EGF plus eIF5A siRNA-treated groups. The data analysis showed that eIF5A, MMP9, and PCNA expression decreased as a result of the inhibitor LY294002. Apoptotic cells were increased in the EGF plus eIF5A siRNA, EGF plus LY294002, and EGF plus eIF5A siRNA plus LY294002 groups as compared with the EGF siRNA group. CONCLUSIONS: These results indicate that EGF-induced upregulation of eIF5A stimulates corneal epithelial cell proliferation in vitro. EGF stimulation of corneal epithelial proliferation was through the phosphatidylinositol 3-kinase (PI3-k)/protein kinase B (Akt) signaling pathway.
Assuntos
Córnea/citologia , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/citologia , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Apoptose , Proliferação de Células , Ativação Enzimática , Citometria de Fluxo/métodos , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
BACKGROUND: Decay-accelerating factor (DAF) is one of the key molecules involved in cell protection against autologous complement, which restricts the action of complement at critical stages of the cascade reaction. The effect of DAF on the survival of human cervical cancer cell (ME180) has not been demonstrated. METHODS: In this study we applied, for the first time, small interference RNA (siRNA) to knock down the expression of the DAF with the aim of exploiting complement more effectively for tumor cell damage. Meanwhile, we investigated the effects of DAF on the viability and migration, moreover the proliferation of ME180 cell. RESULTS: The results showed that the expression of DAF was significantly increased in human cervical cancer tissues. SiRNA inhibition of DAF expression enhanced complement-dependent cytolysis up to 32% in ME180 cells, which contributed to the control of C3 activation and increased the cells viability, migration and augment the number of ME180 cells. CONCLUSION: These data indicated that DAF siRNA described in this study may offer an additional alternative to improve the efficacy of antibody- and complement-based cancer immunotherapy.
Assuntos
Antígenos CD55/fisiologia , Neoplasias do Colo do Útero/metabolismo , Antígenos CD55/genética , Antígenos CD55/metabolismo , Movimento Celular , Proliferação de Células , Sobrevivência Celular/genética , Convertases de Complemento C3-C5/metabolismo , Regulação para Baixo , Feminino , Humanos , RNA Interferente Pequeno/metabolismo , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
BACKGROUND: Decay-accelerating factor (DAF) and membrane cofactor protein (MCP) are the key molecules involved in cell protection against autologus complement, which restricts the action of complement at critical stages of the cascade reaction. The cooperative effect of DAF and MCP on the survival of human cervical cancer cell (ME180) has not been demonstrated. METHODS: In this study we applied, for the first time, short hairpin RNA (shRNA) to knock down the expression of the DAF and MCP with the aim of exploiting complement more effectively for tumor cell damage. Meanwhile, we investigated the cooperative effects of DAF and MCP on the viability and migration, moreover the proliferation of ME180 cell. RESULTS: The results showed that shRNA inhibition of DAF and MCP expression enhanced complement-dependent cytolysis (CDC) up to 39% for MCP and up to 36% for DAF, and the combined inhibition of both regulators yielded further additive effects in ME180 cells. Thus, the activities of DAF and MCP, when present together, are greater than the sum of the two protein individually. CONCLUSION: These data indicated that combined DAF and MCP shRNA described in this study may offer an additional alternative to improve the efficacy of antibody-and complement-based cancer immunotherapy.
