RESUMO

This study aimed to investigate the mechanism of hypoxia-inducible factor-1 alpha (HIF-1α) mediated hypoxia-induced permeability changes in bladder endothelial cells. Models of in vitro hypoxic cell culture of bladder cancer, bladder cancer cells with low HIF-1α expression and HIF-1α RNA interference (RNAi) expression vector were established. Western blot and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of HIF-1α and vascular endothelial growth factor (VEGF) in each group. Bladder cell permeability was determined. Results showed that protein and mRNA expression of HIF-1α and VEGF at 3 and 12 h of hypoxia were significantly higher than normal control (P<0.05), and peaked at 12 h. HIF-1α and VEGF expression in the hypoxic group and hypoxic+3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) group were significantly higher than normal control (P<0.05), while expression in the hypoxic+YC-1 group was significantly lower than the hypoxic group (P<0.05). Bladder cell permeability in the hypoxic and hypoxic+YC-1 group were significantly increased compared to normal control (P<0.05), while in the hypoxic+YC-1 group was significantly decreased compared to the hypoxic group (P<0.05). Most of the cells in the stably transfected HIF-1α RNAi expression vector pcDNA6.2-GW/EmGFP-miR-siHIF-1α expressed green fluorescence protein (GFP) under fluorescence microscope. pcDNA6.2-GW/EmGFP-miR-siHIF-1α could significantly inhibit HIF-1α gene expression (P<0.05). HIF-1α and VEGF expression in the hypoxic group and siHIF-1α hypoxic group were significantly higher than normal group (P<0.05), while expression in the siHIF-1α hypoxic group was significantly lower than the hypoxic group (P<0.05). Findings suggest that HIF-1α is an important factor in the increase of bladder cancer cell permeability.

Assuntos

Animais , Ratos , Neoplasias da Bexiga Urinária/metabolismo , Células Endoteliais/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Hipóxia Tumoral/fisiologia , Permeabilidade , Regulação Neoplásica da Expressão Gênica/fisiologia , Western Blotting , Interferência de RNA , Linhagem Celular Tumoral , Células Endoteliais/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Reação em Cadeia da Polimerase em Tempo Real

RESUMO

Bufo arenarum eggs at late blastula and gastrula were treated with tunicamycin, an inhibitor of glycoprotein glycosylation, to investigate its effects on morphogenesis and neural induction. Because of the low permeability of the amphibian egg to a number of drugs, the blastocoel was opened surgically prior to treatment. Almost all of the eggs treated with the antimetabolite, at a concentration of 10 µg/ml, from late blastula stage for 24h exhibited exogastrulation. The effect is dose- and stage-dependent as shown by the lower proportion of exogastrulae obtained when eggs are treated at a lower concentration (5 µg/ml) or after the onset of gastrulation. Treatment with the antimetabolite did not interfere with neural induction, as partial exogastrulae developed a small neural tube. The most striking biochemical effect was an enhanced uptake of glucose, mannose and leucine. The incorporation of mannose into acid-insoluble material was severely inhibited by tunicamycin, with a concomitant decrease of leucine incorporation into the acid-soluble pool.