Integrative bioinformatics and experimental validation identify pyrroline-5-carboxylate reductase 1 as a mitochondrial regulator and diagnostic biomarker in osteosarcoma.

Osteosarcoma (OS) is a highly aggressive bone malignancy with limited effective biomarkers and therapeutic strategies. An integrative analysis was conducted combining transcriptomic profiling, mitochondrial-related gene (MRG) screening, and machine learning approaches to uncover novel diagnostic and prognostic markers. Among differentially expressed MRGs, enrichment analyses highlighted key metabolic pathways. Machine learning models consistently identified pyrroline 5-carboxylate reductase 1 (PYCR1) as the most robust biomarker, achieving high diagnostic accuracy across independent cohorts. Elevated PYCR1 expression was significantly associated with poor prognosis. Functional experiments demonstrated that PYCR1 knockdown suppressed OS cell proliferation, migration, and invasion, while inducing mitochondrial structural disruption, increased ROS, and altered membrane potential, underscoring its essential role in mitochondrial homeostasis and tumor aggressiveness. Moreover, immune infiltration analysis revealed PYCR1-driven remodeling of the tumor immune microenvironment. Collectively, our findings establish PYCR1 as a mitochondrial regulator integrating metabolic and immunological mechanisms in OS and highlight its potential as a biomarker and therapeutic target for precision diagnosis and mitochondria-targeted interventions.