Introduction Therapeutic oligonucleotide such as antisense-oligonucleotide (AS-ODNs) has been paid great attention because it can specifically hybridize with mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. Therefore, the reduction of side effects is expected. Rat Sarcoma (Ras) gene is a kind of major oncogene. There are three isoforms K-Ras, N-Ras and H-Ras. K-Ras mutation is frequently observed in cancer cells. In normal cells, Ras protein forms active type and inactive type. The active type Ras induces cell proliferation by binding the small molecule GTP. However, in cancer cells having the gene mutations with multiple patterns, Ras always activates, resulting in continuing to send the cell proliferation signal. We have reported that Schizophyllan (SPG) is complexed with ODN and this complex is recognized by β-glucan receptor Dectin-1, β-glucan receptor. Dectin-1 is known to express on immune cells such as macrophage and dendritic cell, and some cancer cells such as lung cancer. In this study we demonstrated the exploration of the optimal AS-ODN sequence for K-Ras and evaluated the gene expression efficiency by the complex. Method We prepared 78 antisense ODNs for K-RAS (K-AS-ODN). After the transfection to Panc 1 cells, human pancreatic epithelioid carcinoma, we evaluated the cell viability by WST-8 assay. After treatment with K-AS-ODN, the expression level of K-Ras protein was confirmed by Western Blotting. We added SPG/K-AS-ODN complex to Panc1 cells and evaluated the cell viability. Result and Discussion Among 78 K-AS-ODNs, 5 AS-ODNs showed a strong inhibition efficiency for Panc-1 cell growth. Considering a secondary structure of K-Ras mRNA obtained by CentroidFold, the five sequences can be easy to bind to a single chain region. The addition of SPG/K-AS-ODN to the cells suppressed not only the gene expression but the cell proliferation. These results could facilitate the clinical application of the complex for cancer therapy.

Development of nucleic acid delivery system targeting Ras gene by β-glucan

256th National Meeting (2018)

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