Zhixun Yin1, Hongmei Ding2, Erxing He1, Jingchen Chen1, Ming Li1. 1. Department of Orthopaedic Surgery, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China. 2. Department of Anatomy, Guangzhou Medical University, Guangzhou, Guangdong, China.
Abstract
BACKGROUND AND OBJECTIVES: MicroRNAs are small non-coding RNAs involved in pathogenesis and progression of human malignancies. MicroRNA-491-5p (miR-491-5p) is down-regulated in many human cancers where it would serve as a tumour suppressor. However, the role played by miR-491-5p in pathogenesis of human osteosarcoma has remained largely unknown. This study has been conducted to examine effects of miR-491-5p on migration and proliferation of cells of the SAOS-2 and MG63 osteosarcoma lines, and mechanisms of those effects. MATERIALS AND 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. 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.
BACKGROUND AND OBJECTIVES: MicroRNAs are small non-coding RNAs involved in pathogenesis and progression of humanmalignancies. MicroRNA-491-5p (miR-491-5p) is down-regulated in many humancancers where it would serve as a tumour suppressor. However, the role played by miR-491-5p in pathogenesis of humanosteosarcoma has remained largely unknown. This study has been conducted to examine effects of miR-491-5p on migration and proliferation of cells of the SAOS-2 and MG63 osteosarcoma lines, and mechanisms of those effects. MATERIALS AND METHODS: Levels of miR-491-5p expression in osteosarcoma tissues and in humanosteosarcoma 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 FOXP4oligonucleotides 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. CONCLUSION: Our in vitro studies showed that up-regulation of miR-491-5p suppressed proliferation of the humanosteosarcoma 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 humanosteosarcoma.
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