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ObjectiveOsteonecrosis 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.MethodsMicroarray 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.ResultsAfter 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 in vitro 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.ConclusionAPOD 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.

Colon cancer (CC) is one of the leading causes of cancer related mortality. Research over past decades have profoundly enhanced our understanding of immunotherapy, a major clinical accomplishment, and its potential role toward treating CC. However, studies investigating the expression of these immune checkpoints, such as epithelial cell adhesion molecule (EpCAM), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1), by peripheral blood mononuclear cells (PBMCs) is lacking. Here, high-dimensional mass cytometry (CyTOF) is used to investigate immune alterations and promising immunotherapeutic targets expression by PBMCs of CC patients. Results reveal that expression of EpCAM and PD-L1 on CD4+ T cells significantly increased in patients with CC, compared with age- and sex- matching healthy controls and patients with colonic polyps. These differences are also validated in an independent patient cohort using flow cytometry. Further analysis revealed that EpCAM+ CD4+ T cells are PD-L1+ CCR5+ CCR6+. Immunofluorescence staining results demonstrate that the increase of EpCAM+ CD4+ T cells is also observed in tumor tissues, rather than para-cancerous tissues. To ascertain the functional disorders of the identified cell subset, phosphorylated signaling protein levels are assessed using imaging mass cytometry. Increases in pp38 MAPK and pMAPKAPK2 are observable, indicating abnormal activation of pp38 MAPK-pMAPKAPK2 signaling pathway. Results in this study indicate that EpCAM+ CD4+ T cells may play a role in CC development. Detailed knowledge on the functionality of EpCAM+ CD4+ T cells is of high translational relevance.

BackgroundRenal clear cell carcinoma (ccRCC) is one of the most prevalent cancers worldwide. Accumulating evidence revealed that copper-induced cell death played a vital role in various tumors. However, the underlying mechanism of cuproptosis with molecular heterogeneity and tumor microenvironment (TME) in ccRCC remains to be elucidated. The present study aimed to discover the biological function of cuproptosis regulators with the potential to guide clinical therapy.MethodsUsing Single-cell RNA-seq, bulk transcriptome and other multi-omics datasets, we identify essential cuproptosis-related hub gene PDHB for further study. The dysregulation of PDHB in ccRCC was characterized, together with survival outcomes, pathway enrichment and immune infiltration among tumor microenvironments. The functional significance and clinical association of PDHB was validated with loss of function experiments and surgical removal specimens.ResultsPDHB mRNA and protein expression level was significantly downregulated in ccRCC tissues compared with normal and paired normal tissues. Clinicopathological parameters and tissue microarray (TMA) indicated that PDHB was identified as a prognostic factor for survival outcomes among ccRCC patients. Additionally, low PDHB was negatively correlated with Treg cells, indicating an immunosuppressive microenvironment. Mechanistically, knockdown PDHB appeared to promote the RCC cells proliferation, migration, and invasion potentials. Subsequent studies showed that copper-induced cell death activation could overcome sunitinib resistance in RCC cells.ConclusionThis research illustrated a cuproptosis-related hub gene PDHB which could serve as a potential prognostic marker and provide therapeutic benefits for clinical treatment of ccRCC patients.