Utilizing bioinformatics to identify biomarkers and analyze their expression in relation to immune cell ratios in femoral head necrosis.

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:40308566", "kind": "science-record", "revision": 3, "content_hash": "sha256:faab68ae51f3eef6c65aac6d90b6d61fac0077243b51ee79e30bb13a7b870fb1", "created_at": "2026-11-01T20:30:27Z", "updated_at": "2026-11-01T22:13:59Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "Utilizing bioinformatics to identify biomarkers and analyze their expression in relation to immune cell ratios in femoral head necrosis.", "doi": "10.3389/fphys.2025.1373721", "pmid": "40308566", "authors": ["Dongchen Li", "Zhilong Huang", "Teng Ma", "Yu Su", "Zhao Li", "Liang Sun", "Ming Li", "Zhong Li", "Yao Li", "Qian Wang", "Yao Lu"], "journal": "Frontiers in physiology", "year": 2025, "abstract": "Background: Necrosis of the Femoral Head (NFH) represents a challenging orthopedic condition, characterized by elusive early detection and rapid progression, predominantly in the middle-aged demographic. Current research on the pathophysiological and immunoregulatory mechanisms underpinning immune cell infiltration in NFH is sparse. This study employs bioinformatics analysis of publicly available RNA sequencing databases to elucidate the pivotal molecules and pathways implicated in NFH progression.\nMethods: The NFH-related dataset GSE123568 was obtained from the Gene Expression Omnibus (GEO). Subsequently, CIBERSORT was utilized to assess the proportion and distribution of immune cell types, followed by the identification of critical Hub immune cells using LASSO and RFE algorithms. The dataset GSE123568 was then explored to identify significantly differentially expressed genes (DEGs). These genes were further refined by intersecting with death-associated genes reported in existing literature. GO and KEGG pathway enrichment analyses were conducted to elucidate their underlying molecular mechanism. A protein-protein interaction (PPI) network was constructed using the STRING database and visualized via Cytoscape. Hub genes were identified using the CytoHubba plugin, followed by enrichment analysis, and their expression levels were evaluated using the ROC curve. In addition, we performed expression data visualization and ROC curve analysis on the external dataset GSE74089 to further evaluate the discriminative power of the hub genes. Moreover, the study analyzed the correlation between the identified hub genes and Hub immune cells. Finally, we verified the hub genes utilizing real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry.\nResults: Four types of 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 the findings were validated using the external dataset GSE74089. Among these, STAT3 exhibited the most pronounced positive correlation with neutrophils (r = 0.6804, p = 3.525e-05). Conversely, XIAP displayed the most significant negative correlation with Myeloid dendritic cell activated (r = -0.3610, p = 0.04003). In experiments, the experimental outcomes for five hub genes (CASP8, TNFRSF1A, AKT1, XIAP and STAT3) were congruent with the results obtained from bioinformatics analysis.\nConclusion: Our study identified CASP8, TNFRSF1A, AKT1, XIAP, STAT3 and BCL2L1 as potential biomarkers for NFH patients and elucidated the immune cell types with the strongest association to these markers. These insights may be crucial for the early diagnosis, understanding of the pathophysiological mechanisms, and the development of treatment strategies for NFH.", "claims": [{"id": "claim-method", "statement": "Wilcoxon rank sum test was used to compare the differences in the distribution of different immune cells between NFH and CTRL.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:40308566", "locator": "sections[14] \"Statistical analysis\" paragraph 2", "quote": "Wilcoxon rank sum test was used to compare the differences in the distribution of different immune cells between NFH and CTRL."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:40308566", "locator": "sections[28] \"Data availability statement\" paragraph 2", "quote": "The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material."}], "state": "complete"}, {"id": "claim-target-result", "statement": "Using CIBERSORT-inferred immune-cell proportions from GSE123568 and a Wilcoxon rank sum test, the study identified four hub immune cell types: Neutrophil, Mast cell resting, Myeloid dendritic cell activated, and Macrophage M0.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:40308566", "locator": "sections[5] \"Identification of hub immune cells\" paragraph 2", "quote": "The GSE123568 dataset served as the training set in this study, and least absolute shrinkage and selection operator (LASSO) and recursive feature elimination (RFE) was used to screen the immune cell features. The procedural methodology was as follows: Initially, the lars package (v1.2) (Goeman, 2010) was instrumental in conducting regression analysis to identify specific combinations of immune cells. Subsequently, the RFE algorithm within the caret package (Deist et al., 2018) was utilized for further screening of these immune cell combinations. Ultimately, the immune cells identified by both algorithms were cross-referenced with those exhibiting significant distributional differences as determined by the Wilcoxon test, with the intersecting subset designated as the hub immune cells."}, {"asset": "source:pmid:40308566", "locator": "sections[17] \"Identification of hub immune cells\" paragraph 2", "quote": "By utilizing the LASSO and RFE algorithms, four immune cell types were identified: Neutrophil, Mast cell resting, Myeloid dendritic cell activated, and Macrophage M0 (Supplementary Table S4)."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "Using CIBERSORT-inferred immune-cell proportions from GSE123568 and a Wilcoxon rank sum test, the study identified four hub immune cell types: Neutrophil, Mast cell resting, Myeloid dendritic cell activated, and Macrophage M0.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:40308566"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. This lane did not download the dataset or read its licence.", "duration_hours": null, "parameters": {}, "inputs": ["data:geo:GSE123568"], "outputs": ["the source data with its repository provenance"], "instruments": [], "reagents": []}, {"id": "run_analysis", "order": 3, "name": "Run the published analysis", "kind": "analysis", "state": "blocked", "blocked_on": "method:group_comparison_wilcoxon", "description": "Apply the paper's statistical method to the resolved series and compare the result with the published target.", "duration_hours": null, "parameters": {}, "inputs": ["data:geo:GSE123568"], "outputs": ["the replicated result and its comparison"], "instruments": [], "reagents": []}], "materials": [], "instruments": [], "reagents": [], "conditions": {}, "gaps": ["step:run_analysis blocked on method:group_comparison_wilcoxon"]}, "data": {"state": "partial", "assets": [{"id": "source:pmid:40308566", "role": "manuscript", "format": "xml", "media_type": "application/xml", "uri": "https://doi.org/10.3389/fphys.2025.1373721", "sha256": "sha256:d6db553ade0089a4d9d1b5df583c3c385fe6dea92e7bf26b386d8182027697b3", "bytes": 56341, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "10.3389/fphys.2025.1373721", "version": null, "note": "Full article fetched from PMC; the checksum covers the rendered section body, not the original XML bytes.", "structure": {"pmcid": "PMC12040900", "source": "europe_pmc_fulltext", "coverage": "whole_body", "text_hash_scope": "rendered_section_body"}}, {"id": "data:geo:GSE123568", "role": "raw", "format": "geo-series", "media_type": "application/vnd.ncbi.geo-series+json", "uri": "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE123568", "sha256": null, "bytes": null, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "GSE123568", "version": null, "note": "The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.", "structure": {"repository": "GEO", "access_route": "GEO accession URL", "access_statement_verbatim": "The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.", "access_statement_kind": "data_availability_statement", "sample_accessions": ["GSM3507251", "GSM3507252", "GSM3507253", "GSM3507254", "GSM3507255", "GSM3507256", "GSM3507257", "GSM3507258", "GSM3507259", "GSM3507260", "GSM3507261", "GSM3507262", "GSM3507263", "GSM3507264", "GSM3507265", "GSM3507266", "GSM3507267", "GSM3507268", "GSM3507269", "GSM3507270", "GSM3507271", "GSM3507272", "GSM3507273", "GSM3507274", "GSM3507275", "GSM3507276", "GSM3507277", "GSM3507278", "GSM3507279", "GSM3507280", "GSM3507281", "GSM3507282", "GSM3507283", "GSM3507284", "GSM3507285", "GSM3507286", "GSM3507287", "GSM3507288", "GSM3507289", "GSM3507290"], "sample_count": 40, "group_sizes": null, "group_sizes_basis": null, "platform": "15207", "taxon": "Homo sapiens", "title": "Identification of Potential Biomarkers for Improving the Precision of Early Detection of Steroid-induced Osteonecrosis of the Femoral Head", "resolution_checked_at": "2026-11-01T15:39:54Z", "resolution_check": "identifier_resolution_only_no_download_no_licence_read", "downloaded": false, "licence_read": false}}, {"id": "data:geo:GSE74089", "role": "raw", "format": "geo-series", "media_type": "application/vnd.ncbi.geo-series+json", "uri": "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE74089", "sha256": null, "bytes": null, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "GSE74089", "version": null, "note": "The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.", "structure": {"repository": "GEO", "access_route": "GEO accession URL", "access_statement_verbatim": "The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.", "access_statement_kind": "data_availability_statement", "sample_accessions": ["GSM1909502", "GSM1909503", "GSM1909504", "GSM1909505", "GSM1909506", "GSM1909507", "GSM1909508", "GSM1909509"], "sample_count": 8, "group_sizes": null, "group_sizes_basis": null, "platform": "13497", "taxon": "Homo sapiens", "title": "Gene expression profiling of hip cartilage with necrosis of femoral head", "resolution_checked_at": "2026-11-01T15:39:55Z", "resolution_check": "identifier_resolution_only_no_download_no_licence_read", "downloaded": false, "licence_read": false}}], "gaps": ["asset:data:geo:GSE123568 not downloaded", "asset:data:geo:GSE123568 licence not read", "asset:data:geo:GSE74089 not downloaded", "asset:data:geo:GSE74089 licence not read"]}, "analysis": {"state": "partial", "methods": [{"id": "method-1", "family": "group_comparison", "specific": "wilcoxon", "label": "Group comparison", "dsl_operations": [], "missing_operations": ["group_comparison_wilcoxon"], "expressible": false, "parameters": {}, "data_shapes": ["two_or_more_groups_continuous"], "evidence": {"score": 1.0, "band": "verbatim", "quote": "Wilcoxon rank sum test was used to compare the differences in the distribution of different immune cells between NFH and CTRL."}}, {"id": "method-2", "family": "group_comparison", "specific": "student_t_test", "label": "Group comparison", "dsl_operations": [], "missing_operations": ["student_t"], "expressible": false, "parameters": {}, "data_shapes": ["two_or_more_groups_continuous"], "evidence": {"score": 0.8, "band": "high", "quote": "Student's t-test"}}, {"id": "method-3", "family": "machine_learning", "specific": "support_vector_machine", "label": "Machine learning", "dsl_operations": ["train_classifier", "cross_validate", "roc_auc"], "missing_operations": [], "expressible": true, "parameters": {}, "data_shapes": ["labelled_feature_matrix"], "evidence": {"score": 0.8, "band": "high", "quote": "SVM"}}, {"id": "method-4", "family": "correlation_analysis", "specific": "pearson", "label": "Correlation analysis", "dsl_operations": ["pearson_r"], "missing_operations": [], "expressible": true, "parameters": {}, "data_shapes": ["paired_numeric_columns"], "evidence": {"score": 0.8, "band": "high", "quote": "Pearson correlation"}}, {"id": "method-5", "family": "network_analysis", "specific": "ppi_network", "label": "Network / pathway analysis", "dsl_operations": ["build_graph", "centrality_scores", "pathway_enrich"], "missing_operations": [], "expressible": true, "parameters": {}, "data_shapes": ["edge_list"], "evidence": {"score": 0.7, "band": "medium", "quote": "protein-protein interaction"}}, {"id": "method-6", "family": "network_analysis", "specific": "pathway_enrichment", "label": "Network / pathway analysis", "dsl_operations": ["pathway_enrich"], "missing_operations": [], "expressible": true, "parameters": {}, "data_shapes": ["edge_list"], "evidence": {"score": 0.7, "band": "medium", "quote": "pathway enrichment"}}, {"id": "method-7", "family": "diagnostic_evaluation", "specific": "roc", "label": "Diagnostic test evaluation", "dsl_operations": ["confusion_counts", "roc_curve", "sensitivity_specificity", "solve_confusion_matrix", "classification_metrics", "published_precision_envelope"], "missing_operations": [], "expressible": true, "parameters": {}, "data_shapes": ["reference_standard_and_test"], "evidence": {"score": 0.7, "band": "medium", "quote": "ROC curve"}}], "targets": [], "gaps": ["method.group_comparison_wilcoxon"]}, "capability": {"state": "complete", "requirements": [{"capability_id": "capability:computational_analysis", "label": "Computational analysis platform", "assay_families": ["meta_analysis", "machine_learning", "survival_analysis", "statistical_regression", "pca", "molecular_docking", "network_analysis", "differential_expression", "gwas"], "availability_state": "owned", "tier_unlocked": "R1", "candidate": "Automated Assays Laravel application and simulated adapters"}], "gaps": []}, "readiness": {"state": "complete", "tier": "R1", "blocking_axis": "none", "blocked_on": "nothing:replicable_now", "axes": {"method": {"tier": "R1", "expressible": false, "required_operations": [], "missing_operations": ["group_comparison_wilcoxon"], "basis": "The paper names the method it used and the corpus's method extractor maps it onto the DSL catalogue: wilcoxon."}, "capability": {"tier": "R1", "capability_id": "capability:computational_analysis", "availability_state": "owned", "basis": "The computational analysis is owned. The physical bench the paper used is not, so a physical validation cannot be run here."}, "data": {"tier": "R1", "reachable": true, "accessions": ["GSE123568", "GSE74089"], "resolved_accessions": ["GSE123568", "GSE74089"], "statements": ["The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material."], "basis": "The paper names GSE123568, GSE74089 in NCBI GEO database and the accession resolves in the repository. No dataset was downloaded and no licence was read."}}, "can_run": {"dsl_operations": [], "assets": ["source:pmid:40308566", "data:geo:GSE123568"], "steps": []}, "must_build": {"dsl_operations": [{"operation": "group_comparison_wilcoxon", "unlocks_papers": null, "priority": null}], "converters": [], "readers": [], "capabilities": [], "data_gaps": ["data:geo:GSE123568"]}, "gaps": []}, "review": {"state": "partial", "machine_critique": {"kind": "extraction_review", "reader": {"model": "MiniMax-M3.1-Flash", "provider": "minimax", "provider_family": "minimax", "read_at": "2026-11-01T19:51:35Z", "independence": "single_reader"}, "evidence_check": {"method": "verbatim", "availability": "verbatim"}, "calls": 5, "usage": {"prompt_tokens": 16291, "completion_tokens": 1720, "cached_tokens": 256}}, "human_critique": null, "stages": [{"id": "extraction", "state": "complete", "reader": "MiniMax-M3.1-Flash", "provider": "minimax"}], "gaps": ["review.independent_reader", "review.machine_critique", "review.human_critique"]}, "execution": {"state": "absent", "status": "not_run", "mode": null, "runs": [], "gaps": ["execution.not_run"]}, "provenance": {"entities": [{"id": "pmid:40308566", "type": "paper", "label": "pmid:40308566"}, {"id": "source:pmid:40308566", "type": "manuscript", "label": "PMID 40308566 full text"}, {"id": "data:geo:GSE123568", "type": "dataset", "label": "GEO GSE123568"}], "activities": [{"id": "activity-reading-sci-2026-11-01", "type": "classification", "used": [{"id": "pmid:40308566", "type": "paper", "label": "pmid:40308566"}, {"id": "source:pmid:40308566", "type": "manuscript", "label": "PMID 40308566 full text"}, {"id": "data:geo:GSE123568", "type": "dataset", "label": "GEO GSE123568"}], "agent": "agent:corpus-assay-engine"}, {"id": "activity-read-40308566", "type": "extraction", "used": ["source:pmid:40308566"], "agent": "agent:minimax-reader"}, {"id": "activity-resolve-GSE123568", "type": "accession_resolution", "used": ["data:geo:GSE123568"], "agent": "agent:corpus-assay-engine"}], "agents": [{"id": "agent:corpus-assay-engine", "type": "software", "label": "Corpus assay engine"}, {"id": "agent:minimax-reader", "type": "software", "label": "MiniMax literature reader"}], "source_run_id": "reading-sci-2026-11-01", "dsl_catalogue_fingerprint": "343fb30756ba6ec3", "capability_table_fingerprint": "2cb3e42f047d2ecf", "snapshot_id": "ba34b49490d9bfaf"}, "lineage": {"parent_id": "pmid:40308566", "parent_hash": "sha256:6956fc5ca6e420f22b9650a8949fdbae0b8e4568f98a80b79d249b22e5b47fa0", "fork_of": null, "generation": 2}, "gaps": ["asset:data:geo:GSE123568 licence not read", "asset:data:geo:GSE123568 not downloaded", "asset:data:geo:GSE74089 licence not read", "asset:data:geo:GSE74089 not downloaded", "execution.not_run", "method.group_comparison_wilcoxon", "review.human_critique", "review.independent_reader", "review.machine_critique", "step:run_analysis blocked on method:group_comparison_wilcoxon"], "assays": [{"role": "data_generation", "definition_id": "qpcr", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["ence quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. Results: Four typ", "e quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. Results: Four typ"], "order_basis": "paper_text_order:1856", "id": "assay:1"}, {"role": "data_generation", "definition_id": "ihc", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["tive polymerase chain reaction (RT-qPCR) and immunohistochemistry. Results: Four types of immune cells (Neutro", "biomarkers expression levels in qRT-PCR and immunohistochemistry results were analyzed using the independent"], "order_basis": "paper_text_order:1869", "id": "assay:2"}, {"role": "analysis", "definition_id": "group_comparison", "definition_version": "f2132e96ae6c7a4e", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"dsl_catalogue": "f2132e96ae6c7a4e"}, "confidence": 1.0, "evidence": ["Wilcoxon rank sum test was used to compare the differences in the distribution of different immune cells between NFH and CTRL."], "order_basis": "consumes_earlier_output", "id": "assay:3"}]}