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MicroRNAs (miRNAs) are vital gene regulators, which play a profound role in the process of forming and developing many diseases, especially tumour. The study intends to excavate the potential regulatory mechanisms of miR-557 and its targeting gene Homeobox B9 (HOXB9) in osteosarcoma. GEO dataset on osteosarcoma was applied to detect the expression of miR-557 and HOXB9. Associations between miR-557 and HOXB9 were speculated by prediction software and verified by dual luciferase assay. Cell proliferation, colony formation and mobility we