Conclusion
Our in vitro studies showed that up-regulation of miR-491-5p suppressed proliferation of the human osteosarcoma cells and induced apoptosis by targeting FOXP4. These findings suggest that miR-491-5p could be further studied as a potential clinical diagnostic or predictive biomarker for human osteosarcoma.
Methods
Levels of miR-491-5p expression in osteosarcoma tissues and in human osteosarcoma cell lines were studied using qualitative real-time polymerase chain reaction (qRT-PCR) methods. Cell viability was detected using the CCK-8 and EdU assays, while the transwell assay was used to evaluate migration and invasion. Apoptosis was analysed uing flow cytometry and the Hoechst 33342 nuclear staining method. A dual-luciferase reporter system was used to confirm the target gene of miR-491-5p. The electrophoretic mobility shift assay (EMSA) with DIG-labelled double-stranded FOXP4 oligonucleotides was used to confirm whether or not miR-491-5p suppressed FOXP4 activation.
Results
Cells of osteosarcoma tissues and cell lines had low levels of miR-491-5p expression, but high levels of forkhead-box P4 (FOXP4) expression. Transfection of SAOS-2 and MG63 cells with miR-491-5p mimics inhibited expression of FOXP4 protein, which suppressed cell growth and migration, but induced apoptosis. Dual-luciferase reporter assays confirmed FOXP4 as the target gene for miR-491-5p. Overexpression of miR-491-5p suppressed FOXP4 activity in SAOS-2 and MG63 cells. Knockdown of FOXP4 in SAOS-2 and MG63 cells using an RNAi strategy resulted in reduced levels of cell proliferation and migration, but increased levels of apoptosis.