ABSTRACT: Background: Human immunodeficiency virus (HIV)-associated Acquired immunodeficiency syndrome (AIDS), a global public health challenge, is managed by Highly active antiretroviral therapy, though long-term use is linked to chronic complications. Osteonecrosis of the femoral head (ONFH), a prevalent HIV-associated disorder with complex etiology, remains mechanistically unclear. Single-cell RNA sequencing (scRNA-seq) offers novel insights into HIV-ONFH pathogenesis. Methods: In this study, femoral head tissue and bone marrow samples from HIV-ONFH and femoral neck fracture patients underwent single-cell sequencing. Data processing involved Seurat for quality control, identification of highly variable genes, dimensionality reduction, clustering, and cell annotation. Enrichment analyses utilized ReactomeGSEA and gene set variation analysis (GSVA), while key genes were selected via protein-protein interaction (PPI) network construction with CytoHubba algorithms. Subsequent analyses included key gene characterization, molecular regulatory network construction, drug prediction, molecular docking, and single-cell expression profiling via Seurat and Monocle. Results: Key cell types in ONFH were identified, with CD8+ T cells as critical effectors. Single-cell analysis revealed their heterogeneity and communication patterns. Twenty-nine candidate genes were prioritized via PPI network analysis, enriched in pathways like MAPK signaling. Four hub genes (HNRNPU, RPS4Y1, RPS9, RPL35) were validated with chromosomal (Chr1, 9, 19, Y) and subcellular localizations (nucleus, extracellular, cytoplasm), exhibiting ribosomal/physical interaction functions. Molecular regulatory network analysis identified 28 key transcription factors (e.g., hsa-miR-3142, AL_355075_4) driving cell-type-specific modules. Drug-docking simulations revealed high-affinity interactions (CYCLOHEXIMIDE w/RPL35/RPS9: -5.6/-6.6 kcal/mol; ATALUREN w/RPS4Y1: -7.1 kcal/mol). Gender-specific expression patterns and differentiation-dependent trends were observed, with ETS2/CEBPA identified as master regulators in CD8+ subsets. Conclusion: This study analyzed targeted therapeutic genes and molecular mechanisms of HIV-ONFH, linked CD8+ T cell-related genes to the disease, and provided a scientific basis for precise interventions. However, results require further validation by subsequent research.
