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

BACKGROUND

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

METHODS

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.

RESULTS

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.

CONCLUSION

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.