Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. Accumulating evidence has revealed that microRNAs (miRNAs) play a crucial role in the progression of OS. In this study, we found that miR-744-5p was the least expressed miRNA in patients with OS by analyzing GSE65071 from the GENE EXPRESSION OMNIBUS (GEO) database. Through real-time quantitative PCR (qRT-PCR), western blotting, colony formation assay, 5-Ethynyl-2-Deoxyuridine (EdU) incorporation assay, transwell migration, and invasion assays, we demonstrated its ability to inhibit the proliferation, migration, and invasion of OS cells in vitro . According to the luciferase reporter assay, transforming growth factor-β1 (TGFB1) was negatively regulated by miR-744-5p and reversed the effects of miR-744-5p on OS. Subcutaneous tumor-forming animal models and tail vein injection lung metastatic models were used in animal experiments, and it was found that miR-744-5p negatively regulated tumor growth and metastasis in vivo . Furthermore, rescue assays verified that miR-744-5p regulates TGFB1 expression in OS. Further experiments revealed that the p38 MAPK signaling pathway is involved in the miR-744-5p/TGFB1 axis. Generally, this study suggests that miR-744-5p is a negative regulator of TGFB1 and suppresses OS progression and metastasis via the p38 MAPK signaling pathway.

Background: Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents, and accumulating evidence has revealed that microRNAs (miRNAs) exert a crucial part in the progression of OS. Methods: GSE65071 from the GEO database was analyzed and miR-744-5p was found to be the lowest expressed miRNA. Real-time quantitative PCR (qRT-PCR), Western blotting (WB), colony formation assay, 5-Ethynyl-2-Deoxyuridine (EdU) incorporation assay and Transwell migration and invasion assay were performed to examine the effects of miR-744-5p in vitro, Luciferase-reporter assay was performed to detect the interactions between miR-744-5p and its specific target gene. Subcutaneous tumor-forming animal models and tail vein injection lung metastatic models were conducted in animal experiments to detect the effects of miR-744-5p in vivo. Results: miR-744-5p expression was down-regulated in OS cells and tissues. Higher expression of miR-744-5p was related with better clinical prognosis and lower malignancy degree of OS, including cell proliferation, migration and invasion in vitro and vivo. Transforming growth factor-β1 (TGFB1) was negatively regulated by miR-744-5p and could reverse the effects of miR-744-5p on OS proliferation, migration and invasion. The MAPK/ERK signaling pathway was involved in the miR-744-5p/TGFB1 axis. Conclusions: In general, this study suggests that miR-744-5p is a negative regulator of TGFB1, and suppresses OS progression and metastasis via MAPK/ERK signaling pathway.

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