Background
Osteosarcoma progression is a major determinant of poor clinical outcome, yet molecular regulators that reliably reflect disease advancement remain insufficiently defined. Guide miRNA strands are important for regulating gene expression after transcription, while the accompanying passenger strands are generally considered non-functional and less researched. The aim was to systematically analyze miR-16-1-3p, a passenger strand derived from the miR-16-1 gene, to identify target genes that are prognostically significant in OS progression.

Methods
Transcriptomic and clinical data from the TARGET-OS cohort were analyzed to investigate the association between miR-16-1-3p regulatory activity and osteosarcoma progression. miR-16-1-3p activity was inferred indirectly through expression patterns of its predicted target gene set using gene set enrichment analysis and cumulative Z-score–based scoring. Associations with progression status and clinical outcomes were assessed. To validate intrinsic miR-16-1-3p target binding activity in living cells, a fluorescent miRNA sensor system was developed. Functional impact of miR-16-1-3p targeting was further examined using proliferation, migration and cisplatin sensitivity.

Results
The miR-16-1-3p target gene set was significantly enriched in patients with progressive disease, and higher cumulative target gene expression scores were associated with unfavorable clinical outcomes. The fluorescent sensor system directly confirmed that miR-16-1-3p recognizes and suppresses complementary target sequences, demonstrating bona fide regulatory activity. Functional assays showed that miR-16-1-3p overexpression suppressed multiple progression-associated malignant phenotypes and reduced tumor growth and metastasis in the CAM model.

Conclusion
These findings identify miR-16-1-3p as a functionally active passenger strand whose regulatory activity is associated with OS progression. The study effectively merges clinical transcriptomic analysis with sensor validation and functional models, underscoring the vital role played by miRNA passenger strands in disease progression and presenting innovative insights into miRNA regulation in OS.