Department of Bone and Joint Surgery, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
Front Immunol. 2024 Jan 11;14:1340446. doi: 10.3389/fimmu.2023.1340446. eCollection 2023.
Osteonecrosis of the femoral head (ONFH) is a common orthopedic condition that will prompt joint dysfunction, significantly impacting patients' quality of life. However, the specific pathogenic mechanisms underlying this disease remain elusive. The objective of this study is to examine the differentially expressed messenger RNAs (DE mRNAs) and key genes linked to ONFH, concurrently investigating the immune cell infiltration features in ONFH patients through the application of the CIBERSORT algorithm.
Microarray was applied to scrutinize mRNA expression profiles in both ONFH patients and healthy controls, with data integration sourced from the GEO database. DE mRNAs were screened using the Limma method. The biological functions of DE mRNAs were explored through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, Gene Ontology (GO) functional analysis, and Gene Set Enrichment Analysis (GSEA). Additionally, support vector machine-recursive feature elimination (SVM-RFE) and the least absolute shrinkage and selection operator (LASSO) were employed to discern diagnostic biomarkers associated with the disease. Receiver operating characteristic (ROC) analysis was utilized to assess the statistical performance of the feature genes. The validation of key genes was performed using qRT-PCR in bone tissues obtained from ONFH patients and healthy controls. Osteogenic differentiation of BMSC was then performed and detected by alkaline phosphatase staining (ALP) and qRT-PCR to verify the correlation between key genes and osteogenic differentiation. Finally, immune cell infiltration analysis was executed to evaluate immune cell dysregulation in ONFH, concurrently exploring the correlation between the infiltration of immune cells and key genes.
After consolidating the datasets, the Limma method revealed 107 DEGs, comprising 76 downregulated and 31 upregulated genes. Enrichment analysis revealed close associations of these DE mRNAs with functions such as cell migration, osteoblast differentiation, cartilage development and extracellular region. Machine learning algorithms further identified APOD, FBXO43 and LRP12 as key genes. ROC curves demonstrated the high diagnostic efficacy of these genes. The results of qRT-PCR showed that the expression levels of key genes were consistent with those of microarray analysis. In addition, the results of experiments showed that APOD was closely related to osteogenic differentiation of BMSC. Immune infiltration analysis suggested a close correlation between ONFH and imbalances in levels of Neutrophils, Monocytes, Macrophages M2, Dendritic cells activated and Dendritic cells resting.
APOD is closely related to osteogenic differentiation of BMSCs and can be used as a diagnostic marker of ONFH. Immune cell infiltration significantly differs between controls and ONFH patients.
股骨头坏死（ONFH）是一种常见的骨科疾病，会导致关节功能障碍，严重影响患者的生活质量。然而，这种疾病的确切发病机制仍不清楚。本研究的目的是通过 CIBERSORT 算法研究与 ONFH 相关的差异表达信使 RNA（DE mRNAs）和关键基因，并探讨 ONFH 患者的免疫细胞浸润特征。
使用微阵列分析 ONFH 患者和健康对照组的 mRNA 表达谱，数据整合自 GEO 数据库。使用 Limma 方法筛选差异表达基因。通过京都基因与基因组百科全书（KEGG）通路富集分析、基因本体论（GO）功能分析和基因集富集分析（GSEA）探讨 DE mRNAs 的生物学功能。此外，采用支持向量机递归特征消除（SVM-RFE）和最小绝对值收缩和选择算子（LASSO）筛选与疾病相关的诊断生物标志物。使用接收器工作特征（ROC）分析评估特征基因的统计性能。使用 qRT-PCR 在取自 ONFH 患者和健康对照组的骨组织中验证关键基因。然后进行骨髓间充质干细胞（BMSC）成骨分化实验，并通过碱性磷酸酶染色（ALP）和 qRT-PCR 进行检测，以验证关键基因与成骨分化的相关性。最后，进行免疫细胞浸润分析，以评估 ONFH 中的免疫细胞失调，并探讨免疫细胞浸润与关键基因的相关性。
整合数据集后，Limma 方法发现 107 个 DEGs，包括 76 个下调基因和 31 个上调基因。富集分析表明，这些 DE mRNAs 与细胞迁移、成骨细胞分化、软骨发育和细胞外区等功能密切相关。机器学习算法进一步鉴定出 APOD、FBXO43 和 LRP12 为关键基因。ROC 曲线表明这些基因具有较高的诊断效能。qRT-PCR 结果表明，关键基因的表达水平与微阵列分析结果一致。此外，实验结果表明，APOD 与 BMSC 的成骨分化密切相关。免疫浸润分析表明，ONFH 与中性粒细胞、单核细胞、M2 巨噬细胞、激活树突状细胞和静止树突状细胞水平失衡密切相关。
APOD 与 BMSCs 的成骨分化密切相关，可作为 ONFH 的诊断标志物。免疫细胞浸润在对照组和 ONFH 患者之间存在显著差异。
