The Analysis of Differentially Expressed circRNAs Under the Antiproliferative Effect From 5-Fluorouracil on Osteosarcoma Cells

5-fluorouracil (5-FU) is a widely used drug for cancer treatment, but its effect and underlying mechanisms on osteosarcoma (OS) cells remain unclear.

5-FU treatment resulted in the differentially expressed circRNAs that were proliferation- and apoptosis-associated and were involved in the 5-FU-induced inhibition of tumor proliferation in OS cells.

U2OS and MG63 cells were treated with 0, 50, 100, and 500 μM 5-FU. MTS and flow cytometry were used to examine the effect of 5-FU on cell viability and apoptosis, respectively. Circular RNA (circRNA) expression was detected using RNA sequencing and quantitative real-time PCR (qPCR). Differentially expressed circRNAs were further subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) analysis to predict their functions. A circRNA-miRNA-mRNA interaction network was generated to analyze the regulatory networks of 5-FU-induced differentially expressed circRNAs. Western blotting (WB) was used to verify the protein in the downstream of circRNAs.

5-FU inhibited the cell viability of the MG63 cells in a concentration-dependent manner. The most significant effect was observed in the cells treated with 500 μM 5-FU. Apoptosis was also increased in the MG63 cells after 500 μM 5-FU treatment for 3 days. RNA sequencing results showed that 183 differentially expressed circRNAs (172 upregulated and 11 downregulated) in 5-FU-treated cells. KEGG and GO analysis showed that the differentially expressed circRNAs were primarily enriched in proliferation-, apoptosis-, and metabolism-related functions. qPCR was used to verify the most upregulated and downregulated circRNAs. The circRNA-miRNA-mRNA interaction network showed that these 8 circRNAs had a sizable regulatory network that links a series of genes involved in tumor suppression.