Utilizing Bioinformatics to Identify Biomarkers and Analyz e Their Expression in Relation to Immune Cell Ratios in Fe moral Head Necrosis

Dongchen Li¹Zhilong Huang²Teng Ma¹Yu Su¹Zhao Li¹Liang Sun¹Ming Li¹Zhong Li¹Yao Li^1*Qian Wang^1*Yao Lu^3*

• ¹Xi'an Honghui Hospital, Xi'an, Shaanxi Province, China
• ²The Nuclear Industry 417 Hospital,, Xi’an, China
• ³Xi'an Jiaotong University, Xi'an, China

Background: Necrosis of the Femoral Head (NFH) is a challenging orthopedic condition, characterized by difficult early detection and rapid progression, particularly in middle-aged individuals. Research on the pathophysiological and immunoregulatory mechanisms of immune cell infiltration in NFH is limited. This study uses bioinformatics analysis of publicly available RNA sequencing databases to identify key molecules and pathways involved in NFH progression.Methods: The NFH-related dataset GSE123568 was retrieved from the Gene Expression Omnibus (GEO). CIBERSORT was employed to assess immune cell proportions and distribution, followed by the identification of critical hub immune cells using LASSO and RFE algorithms. Differentially expressed genes (DEGs) were identified and intersected with death-associated genes in existing literature. GO and KEGG pathway enrichment analyses were performed to reveal underlying molecular mechanisms. A protein-protein interaction (PPI) network was constructed via the STRING database and visualized in Cytoscape. Hub genes were identified using the CytoHubba plugin, followed by enrichment analysis, and their expression levels were evaluated using the ROC curve. Additionally, expression data from the external dataset GSE74089 was used to assess the hub genes' discriminative power. Correlations between hub genes and immune cells were analyzed. The findings were validated using RT-qPCR and immunohistochemistry.Results: Four immune cells (Neutrophil, Mast cell resting, Myeloid dendritic cell activated, Macrophage M0) were identified. Fourteen pivotal genes (BCL2L1, BIRC2, NFKBIA, XIAP, CFLAR, AKT1, BIRC3, IKBKB, RIPK1, CASP8, TNFRSF1A, IL1B, CASP1, STAT3) were identified and validated using the external dataset GSE74089. STAT3 showed the strongest positive correlation with neutrophils (r = 0.6804, p = 3.525e-05). In contrast, XIAP had the most significant negative correlation with Myeloid dendritic cells (r = -0.3610, p = 0.04003). Experimental results for five hub genes (CASP8, TNFRSF1A, AKT1, XIAP, and STAT3) were consistent with bioinformatics findings.Conclusion: CASP8, TNFRSF1A, AKT1, XIAP, STAT3, and BCL2L1 were identified as potential biomarkers for NFH and correlated with immune cell types. These findings could aid in early diagnosis, understanding the pathophysiology, and developing treatment strategies for NFH.