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Research Article
* These authors contributed equally
Using public blood transcriptome datasets, this study identified candidate renin-angiotensin system (RAS)-related biomarkers for hypertension. Bioinformatics and machine learning yielded an eight-gene signature tested in an independent blood-based cohort, linking RAS transcriptomic alterations to immune and inflammatory signatures requiring further validation.
This work aimed to identify candidate renin-angiotensin system (RAS)-related blood transcriptomic biomarkers associated with hypertension, construct an externally tested candidate diagnostic model, and validate selected genes in an Ang II-induced endothelial cell model. Two public microarray datasets, GSE75360 and GSE74144, were analyzed. Differential expression analysis was performed using limma, followed by GO/KEGG enrichment, preranked GSEA, CIBERSORT immune infiltration analysis, protein-protein interaction and regulatory network construction, and machine learning-based feature selection using logistic regression and random forest. A logistic regression model based on the selected genes was developed in GSE75360 and externally tested in GSE74144. Experimental validation was performed in Ang II-induced HUVECs using qRT-PCR, western blotting, ELISA, and CST3/FURIN loss- and gain-of-function assays, followed by CCK-8, Transwell, inflammatory, oxidative stress, and endothelial function analyses. In GSE75360, 173 differentially expressed genes were identified, including 18 RAS-related differentially expressed genes. Eight candidate genes, LRP1, CTSD, MTHFR, AUTS2, FURIN, CST3, FCER1G, and TBXAS1, were selected by combined machine learning analyses. Enrichment and immune infiltration analyses indicated that these genes were mainly associated with immune and inflammatory signatures. The eight-gene model showed high discrimination in GSE75360 and retained moderate performance in GSE74144. In Ang II-treated HUVECs, most candidate genes were upregulated at the mRNA level, and CST3, FURIN, and TBXAS1 were further validated at the protein level. CST3 and FURIN modulation altered Ang II-induced endothelial viability, migration, expression of inflammatory/adhesion markers, ROS accumulation, and eNOS/NO-related functional readouts. This study identified candidate RAS-related biomarkers and immune-associated signatures in hypertension and developed an externally tested candidate diagnostic model. The in vitro findings support the functional relevance of CST3 and FURIN in Ang II-induced endothelial responses, warranting further clinical and mechanistic validation.
This study focuses on hypertension, a chronic cardiovascular disease that is prevalent globally1. Hypertension significantly increases the risk of serious health complications, including cardiovascular and cerebrovascular events as well as renal damage. This poses a grave threat to patients' quality of life and longevity, while creating a significant social and economic burden2,3. Currently, the diagnosis and treatment of hypertension mainly rely on blood pressure monitoring and antihypertensive medications. However, because the causes of hypertension are complex and heterogeneous, ....
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Overview of analysis workflow
The overall design of this study’s transcriptomic and machine learning-based analysis is illustrated in Figure 1, encompassing key steps: collection of Renin-Angiotensin System-related genes (RASRGs); screening of RAS-related differentially expressed genes (RASRDEGs) from hypertension datasets; functional enrichment analysis (GO/KEGG/GSEA); immune infiltration analysis (CIBERSORT); construction of protein-protein interaction (PPI) and regulatory networks; machine learning-based key gene selection (logistic regression, random forest [RF]); and evaluation of the hype....
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Cleaning of hypertension datasets
To ensure the reliability of subsequent analyses, datasets GSE75360 and GSE74144 were first subjected to probe annotation and data normalization using the R package limma. The distributions of gene expression values before and after normalization are shown in Supplemental File 1—Supplemental Figure S1A-D, with orange representing hypertension samples and blue representing control samples.
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Hypertension is a complex cardiovascular disorder involving RAS dysregulation, immune-inflammatory activation, vascular injury, and endothelial dysfunction32. In the present study, we first used public blood transcriptomic datasets and machine learning approaches to identify RAS-related candidate biomarkers and to build an externally tested candidate diagnostic model. We then extended these bioinformatics findings through experimental validation in an Ang II-induced HUVEC model. This combined stra.......
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The authors have no conflicts of interest to declare.
Authors' contributions:
Conception and design of the work: Bai J, Wang Y;
Data collection: Yang X X, Du L, Shi J H, Li X X, Bai M;
Supervision: Bai J, Wang Y;
Analysis and interpretation of the data: Yang X X, Du L, Shi J H, Li X X, Bai M;
Statistical analysis: Bai J, Wang Y, Bai M;
Drafting the manuscript: Bai J, Wang Y;
Critical revision of the manuscript: all authors;
Approval of the final manuscript: all authors.
Funding: Gansu Provincial Department of Education Higher Education Faculty Innovation Fund Project (No. 2026B-268)
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 96-well cell culture plates | Used for CCK-8 cell viability assays | ||
| Angiotensin II (Ang II) | Used to induce endothelial injury in HUVECs at 100 nM for 48 h | ||
| Antibiotic solution | Added to endothelial cell culture medium according to the supplier’s instructions | ||
| BCA protein assay kit | Used to determine protein concentration for western blotting | ||
| Cell Counting Kit-8 (CCK-8) | Used to assess HUVEC viability/proliferation | ||
| Chemiluminescence detection reagent | Used to visualize western blot protein bands | ||
| ChIPBase v2.0 | Sun Lab / Research Resource | N/A | Database used to retrieve transcription factor-target relationships for hub genes |
| CIBERSORT | Stanford University / Newman Lab | N/A | Immune cell deconvolution algorithm used to estimate the abundance of 22 immune cell types |
| clusterProfiler (R package) | Bioconductor | N/A | R package used for GO, KEGG, and GSEA analyses |
| CO2 cell culture incubator | Used to culture HUVECs at 37 °C with 5% CO2 | ||
| Comparative Toxicogenomics Database (CTD) | NC State University / Mount Desert Island Biological Laboratory | N/A | Database used to predict gene-drug interactions |
| Complete endothelial cell culture medium | Used for HUVEC culture | ||
| Computer workstation with internet access | Any standard research computing platform | N/A | Used for data download, preprocessing, statistical analysis, and visualization |
| CST3 ELISA kit | Used to detect secreted CST3 in cell culture supernatant | ||
| CST3 overexpression plasmid (oe-CST3) | Used for CST3 gain-of-function experiments in HUVECs | ||
| Cytoscape | Cytoscape Consortium | N/A | Software used to visualize mRNA-miRNA, mRNA-TF, and mRNA-drug regulatory networks |
| DCF fluorescent probe/ROS assay reagent | Used to detect intracellular ROS levels | ||
| Empty-vector control plasmid (oe-NC) | Used as the overexpression control | ||
| Fetal bovine serum | Supplement added to endothelial cell culture medium | ||
| FURIN overexpression plasmid (oe-FURIN) | Used for FURIN gain-of-function experiments in HUVECs | ||
| GeneCards | Weizmann Institute of Science | N/A | Database used to curate RAS-related genes |
| GeneMANIA | University of Toronto | N/A | Web-based tool used to identify functionally related genes |
| GEO database | NCBI | N/A | Public database used to access GSE75360 and GSE74144 datasets |
| GEOquery (R package) | Bioconductor | N/A | R package used to download GEO datasets |
| ggDCA (R package) | CRAN / GitHub source used by authors | N/A | R package used for decision curve analysis |
| ggplot2 (R package) | CRAN | N/A | R package used for data visualization |
| Human umbilical vein endothelial cells (HUVECs) | Cell model used for Ang II-induced endothelial injury experiments | ||
| limma (R package) | Bioconductor | N/A | R package used for normalization and differential expression analysis |
| MSigDB | Broad Institute | N/A | Gene set database used for GSEA |
| Negative control siRNA (si-NC) | Used as the knockdown control | ||
| Nitric oxide assay kit | Used to measure NO levels in culture supernatant | ||
| pheatmap (R package) | CRAN | N/A | R package used to generate heatmaps |
| Primary antibody against CST3 | Used for western blotting | ||
| Primary antibody against FURIN | Used for western blotting | ||
| Primary antibody against GAPDH | Used as the western blot loading control | ||
| Primary antibody against TBXAS1 | Used for western blotting | ||
| pROC (R package) | CRAN | N/A | R package used to generate ROC curves and calculate AUC |
| PubMed | U.S. National Library of Medicine | N/A | Literature database used to supplement RAS-related gene curation |
| PVDF membrane | Used for western blot protein transfer | ||
| qRT-PCR primers | Used to detect candidate genes and inflammatory/endothelial markers | ||
| R software | R Foundation for Statistical Computing | N/A | Statistical computing environment used for all analyses |
| randomForest (R package) | CRAN | N/A | R package used for feature selection by random forest |
| RCircos (R package) | CRAN / Bioconductor-associated resource | N/A | R package used to visualize chromosomal localization of RASRDEGs |
| Reverse transcription kit | Used to synthesize complementary DNA | ||
| RIPA lysis buffer | Used for total protein extraction | ||
| rms (R package) | CRAN | N/A | R package used to construct the nomogram |
| RNA extraction reagent | Used to extract total RNA from HUVECs | ||
| Secondary antibodies | Used for western blotting | ||
| siRNA targeting CST3 (si-CST3) | Used for CST3 knockdown experiments | ||
| siRNA targeting FURIN (si-FURIN) | Used for FURIN knockdown experiments | ||
| starBase v3.0 | Sun Lab / Research Resource | N/A | Database used to identify miRNA-target interactions |
| STRING | STRING Consortium | N/A | Database used to construct the protein-protein interaction network |
| SYBR Green qPCR reagent | Used for qRT-PCR detection | ||
| Transfection reagent | Used to transfect siRNAs and overexpression plasmids into HUVECs | ||
| Transwell chambers | Used for HUVEC migration assays |