| Autor: |
Fujiwara N; Department of Chemistry and Biochemistry, The University of Kitakyushu, 1-1, Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka, 808-0135, Japan., Izumi H; University of Occupational and Environmental Health, 1-1 Isegaoka, Yahatanishi-ku, Kitakyushu, Fukuoka, 807-8555, Japan., Morimoto Y; University of Occupational and Environmental Health, 1-1 Isegaoka, Yahatanishi-ku, Kitakyushu, Fukuoka, 807-8555, Japan., Sakurai K; Department of Chemistry and Biochemistry, The University of Kitakyushu, 1-1, Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka, 808-0135, Japan., Mochizuki S; Department of Chemistry and Biochemistry, The University of Kitakyushu, 1-1, Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka, 808-0135, Japan. mochizuki@kitakyu-u.ac.jp. |
| Abstrakt: |
Antisense oligonucleotides (AS-ODNs) hybridize with specific mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. We previously demonstrated that the β-glucan schizophyllan (SPG) can form a complex with AS-ODNs with attached dA 40 (AS-ODNs/SPG), and this complex can be incorporated into cells, such as macrophages and dendritic cells, expressing the β-glucan receptor Dectin-1. We have achieved efficient gene silencing in animal models, but the uptake mechanism and intracellular distribution are unclear. In this study, we prepared the complex consisting of SPG and AS-ODNs (AS014) for Y-box binding protein-1 (YB-1). After treatment with endocytosis inhibitor Pitstop 2 and small interfering RNA targeting Dectin-1, we found that AS014/SPG complexes are incorporated into cells by Dectin-1-mediated endocytosis and inhibit cell growth in a Dectin-1 expression level-dependent manner. After treatment with AS014/SPG complexes, we separated the cell lysate into endosomal and cytoplasmic components by ultracentrifugation and directly determined the distribution of AS014 by reverse transcription PCR using AS014 ODNs as a template or a reverse transcription primer. In the cytoplasm, AS014 clearly hybridized with YB-1 mRNAs. This is the first demonstration of the distinct distribution of the complex in cells. These results could facilitate the clinical application of the complex. |
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