ENA antisense oligonucleotides for vascular endothelial growth factor (VEGF) mRNA were synthesized and evaluated in A549 lung cancer cells. It was found that the VEGF ENA-antisense inhibited not only the expression of VEGF, but also the expression of three genes, which were found in Genbank by BLAST and Clustal W search and considered likely to bind to the VEGF ENA-antisense. These results indicate that ENA-antisense oligonucleotides act in a sequence-specific manner, and could be used as effective antisense drugs.
We investigated 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) antisense oligonucleotides (AONs) for vascular endothelial growth factor (VEGF) in human lung carcinoma A549 cells. An ENA/DNA gapmer AON with RNase H-mediated activity was virtually stable in rat plasma and exhibited more than 90% inhibition of VEGF mRNA production. Moreover, 22 genes that are likely to bind to the AON were found in the GenBank database by BLAST and CLUSTAL W searches. Three of these genes were actually inhibited by the ENA AON. In shorter ENA AONs with fewer matched sequences of these genes, inhibitiory activities were decreased and off-target effects were improved. These results indicate that ENA AONs act in a sequence-specific manner and could be used as effective antisense drugs.
Mini rats are a transgenic rat strain carrying antisense gene for rat growth hormone (GH), resulting in retarded growth and a lower blood GH level (136 +/- 42.0 ng/mL) compared with that of age-matched parental strain Wistar rats (329 +/- 337 ng/mL). Mini rats have been used by several investigators as a GH deficiency model. In this work, we gave a single oral administration of thioacetamide (TAA), a hepatotoxicant, to both Mini rats and Wistar rats to ascertain the influence of GH deficiency on liver response to chemically induced injury and subsequent regeneration. TAA administration caused liver injury in both strains, with a greater extent of injury in Mini rats. Proliferation of bile epithelial cells and so-called oval cells was prominent at Day 3 in Mini rats only, and this change correlated well with serum total bilirubin concentrations. Antibody against Ki-67 antigen revealed that cellular proliferation after TAA-induced liver injury was suppressed but prolonged in the Mini rat liver. Although hepatic stellate cells and Kupffer cells/macrophages were more abundant in the livers of TAA-treated Mini rats, the hepatic expression patterns of hepatocyte growth factor and transforming growth factor beta 1 were comparable to those of Wistar rats. Insulin-like growth factor-I gene expression was significantly reduced in the Mini rat liver. Our results imply that a lower GH level may exacerbate chemically induced liver injury, enhance infiltration/proliferation of non-parenchymal cells, suppress regeneration of hepatocytes, and induce proliferation of bile epithelial cells and oval cells when the liver is injured by TAA.
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