The miR-16-1-3p passenger strand exhibits functional activity and suppresses malignant phenotypes in osteosarcoma

Background Chemotherapy resistance and disease progression remain major causes of mortality in osteosarcoma (OS). The miR-16 family is linked to tumors, but the specific function and treatment potential of the miR-16-1-3p passenger strand in OS remain unclear. Methods RNA sequencing and clinical data from 82 OS patients in the TARGET-OS cohort were analyzed. Patients were stratified by progression status. Target genes of miR-16-1-3p were predicted using TargetScan and evaluated by gene set enrichment analysis (GSEA). Principal component analysis (PCA) and Kaplan–Meier survival analyses were performed based on cumulative Z-score–derived expression of the target gene set. Functional enrichment was assessed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The impact of lentivirus-transduced miR-16-1-3p on U2OS cells was examined, focusing on proliferation, cell cycle, migration, colony formation, cisplatin sensitivity, and tumor growth in the CAM in vivo model. A fluorescent miRNA sensor was developed to assess intracellular target-binding functionality of the miR-16-1-3p passenger strand. Direct regulation of candidate target gene expression was validated using luciferase reporter assays and quantitative PCR. Expression of validated targets was further examined in a limited cohort of 15 chemotherapy-treated OS patients. Results GSEA demonstrated significant enrichment of predicted miR-16-1-3p target genes among genes upregulated in patients with progressive disease. Kaplan–Meier analysis showed that elevated cumulative expression of predicted miR-16-1-3p target genes was associated with poorer overall survival in osteosarcoma patients. GO and KEGG analyses revealed enrichment of pathways related to translational regulation, mitochondrial function, and chemotherapy-associated signaling. A fluorescent miR-16-1-3p sensor system demonstrated sequence-dependent suppression of reporter activity following miR-16-1-3p overexpression. In functional assays performed in both U2OS and HOS cells, miR-16-1-3p overexpression significantly suppressed proliferation and migration and increased cisplatin sensitivity compared with control (scrambled miRNA) virus-transduced cells. Quantitative PCR and luciferase reporter assays further supported the interaction of miR-16-1-3p with SLC38A1 and ABCA13, two candidate genes potentially associated with chemoresistance. Conclusion The miR-16-1-3p passenger strand exhibited functional activity associated with tumor-suppressive phenotypes and increased cisplatin sensitivity in osteosarcoma cells. Using integrative bioinformatics, fluorescent sensor assays, and functional experiments in U2OS and HOS cells points to miR-16-1-3p possibly regulating cancer characteristics and how osteosarcoma reacts to chemotherapy.