title: Up-regulation of microRNA-491-5p suppresses cell proliferation and promotes apoptosis by targeting FOXP4 in human osteosarcoma.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Orthopaedic Surgery, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.
authors.forename: Zhixun
authors.initials: Z
authors.lastname: Yin
authors.affiliation: Department of Anatomy, Guangzhou Medical University, Guangzhou, Guangdong, China.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Ding
authors.affiliation: Department of Orthopaedic Surgery, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.
authors.forename: Erxing
authors.initials: E
authors.lastname: He
authors.affiliation: Department of Orthopaedic Surgery, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.
authors.forename: Jingchen
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Orthopaedic Surgery, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Li
mesh_terms: D020413:3' Untranslated Regions; D000328:Adult; D000070416:Antagomirs / Q000378:metabolism; D017209:Apoptosis; D001483:Base Sequence; D001859:Bone Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation / Q000235:genetics; D002470:Cell Survival; D005260:Female; D051858:Forkhead Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D016415:Sequence Alignment; D015854:Up-Regulation; D055815:Young Adult
chemical_list: D020413:3' Untranslated Regions; D000070416:Antagomirs; C576732:FOXP4 protein, human; D051858:Forkhead Transcription Factors; D035683:MicroRNAs; D034741:RNA, Small Interfering