Chi3l1 sirna

To determine optimal conditions for forming cell-permeable peptide-siRNA complexes, EGFP or Chi3l1 siRNA (Bioneer) was mixed with dNP2-HA2 peptide (Genscript, KIKKVKKKGRKGSKIKKVKKKGRK-GLFGAIAGFIENGWEGMIDG) which is a combined sequence of a cell-penetrating peptide dNP2 and endosomal escape sequence of HA2^{22 (link)} at various N/P ratios (the number of amine groups in dNP2-HA2 peptide to the number of phosphate groups in siRNA) and incubated for 30 min at RT in the dark. The size of dNP2-siRNA complex was measured by Nano particle size analyzer (K-one).

1.5 μg of Chi3l1 siRNA (Bioneer) was mixed with dNP2-HA2 peptide at various N/P ratios and incubated for 30 min at RT in the dark. After incubation, complexes were mixed with DNA loading dye, and gel electrophoresis was performed on 2% agarose gel for 20 min.

1.25 or 2.5 μg of Chi3l1 siRNA (Bioneer) or EGFP siRNA (Bioneer) was incubated with 35 or 70 μg dNP2-HA2 peptide for 30 min at RT, in the dark. Free siRNA or Peptide-siRNA complexes were intranasally administered to mice, which were sacrificed every 24 h for 3 days. Lung tissue was disrupted in T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific) for protein lysates or in TRIzol for RNA extraction. Chi3l1 protein in the lung was measured by Chi3l1 ELISA kit (R&D Systems). In the lung metastasis model, 5 × 105 B16F10 cells were intravenously injected to WT mice, and then 2.5 μg of siRNA + 70 μg of dNP2-HA2 peptide complex was intranasally administered to anesthetized mice with zoletil + Rompun for twice per day with 10 h interval for every other day until day 14. At 14 days after melanoma injection, mice were sacrificed and analyzed.