Ginsenoside Rg3 inhibits osteosarcoma progression by reducing circ_0003074 expression in a miR-516b-5p/KPNA4-dependent manner

BACKGROUND: Previous data have suggested that ginsenoside Rg3 (Rg3), isolated from the roots of Panax ginseng, plays a repressing role in multiple cancers, including osteosarcoma (OS). However, there is no any literature available about the role of circular RNA (circRNA) in Rg3-mediated OS development. The study aimed to explore the function of circ_0003074 in the anti-cancer effects of Rg3 on OS. METHODS: RNA expression of circ_0003074, miR-516b-5p and karyopherin subunit alpha 4 (KPNA4) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was evaluated by Western blotting or immunohistochemistry assay. Cell viability, proliferation, apoptosis, migration and invasion were investigated by cell counting kit-8, 5-ethynyl-29-deoxyuridine (EdU), flow cytometry analysis, wound-healing and transwell invasion assays, respectively. Dual-luciferase reporter and/or RNA immunoprecipitation assay was performed to confirm the interplay between miR-516b-5p and circ_0003074 or KPNA4. Xenograft mouse model assay was conducted to reveal the effect of Rg3 treatment on tumor formation. RESULTS: Circ_0003074 and KPNA4 expression was significantly upregulated, while miR-516b-5p was downregulated in OS tissues and cells compared with controls. Rg3 treatment dramatically decreased circ_0003074 expression in OS cells. Rg3 treatment led to decreased cell proliferation, migration and invasion but increased cell apoptosis, which was attenuated after circ_0003074 overexpression. Besides, miR-516b-5p was a target miRNA of circ_0003074 and partially restored circ_0003074-mediated action under Rg3 treatment. Decreasing miR-516b-5p expression also promoted Rg3-treated OS cell malignancy through KPNA4, which was identified as a target mRNA of miR-516b-5p. Besides, circ_0003074 induced KPNA4 production owing to the decrease of miR-516b-5p expression. Furthermore, Rg3 treatment inhibited tumor formation by regulating circ_0003074 in vivo. CONCLUSION: Rg3 inhibited OS progression through circ_0003074/miR-516b-5p/KPNA4 axis, showing the potential of Rg3 as a therapeutic agent for OS.