Asparagine-related biomarkers and regulatory mechanisms in type 2 diabetes mellitus

Abstract

Background: Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder. Emerging evidence suggests asparagine metabolism might play a pivotal role in T2DM, yet the underlying molecular mechanisms remain elusive. This study aimed to detect asparagine-related biomarkers and expound their functional roles in T2DM pathogenesis.

Methods: Transcriptomic datasets from peripheral blood samples of T2DM patients and controls were analyzed. Differential expression analysis, protein-protein interaction (PPI) network, and machine learning algorithms, followed by expression analysis across cohorts were employed to screen biomarkers. Biomarker diagnostic performance was evaluated. Functional enrichment, immune infiltration analysis, and multi-layer regulatory network construction were conducted. Drug-target interactions and molecular docking were explored to identify potential therapeutics.

Results: A total of 90 candidate genes were detected. Four feature genes were screened via multi-algorithm integration. Protein phosphatase 1 catalytic subunit alpha (PPP1CA) and cathepsin D (CTSD) were validated as biomarkers, showing significant upregulation in T2DM samples and high diagnostic accuracy (AUC of PPP1CA = 0.969 and CTSD = 0.984 in the training cohort, AUC of PPP1CA = 0.806 and CTSD = 0.875 in the validation cohort, respectively). Functional enrichment highlighted distinct yet complementary functional roles of PPP1CA and CTSD in T2DM progression. Immune infiltration revealed elevated activated dendritic cells, mast cells, and myeloid-derived suppressor cells in T2DM samples, with PPP1CA and CTSD correlating significantly with these cell types. Regulatory networks identified shared transcription factors and miRNAs targeting both genes. Pharmacological screening prioritized norcantharidin and naringenin as high-affinity compounds targeting these biomarkers.

Conclusion: This study identified PPP1CA and CTSD as asparagine-related biomarkers driving immune-metabolic crosstalk in T2DM. The príicted regulatory networks and therapeutic compounds provided novel insights into T2DM mechanisms and potential intervention strategies.

Keywords: asparagine; cathepsin D; immune infiltration; protein phosphatase 1 catalytic subunit alpha; type 2 diabetes mellitus.

FIGURE 1

Identification of candidate genes associated with asparagine in T2DM pathogenesis. (A) Volcano plot of DEGs in GSE15932 (Top 10 marked); (B) Distribution of DEGs in T2DM and healthy control group; (C) Venn plot of DEGs and ARGs; (D) BP analysis of 90 candidate genes; (E) CC analysis of 90 candidate genes; (F) MF analysis of 90 candidate genes; (G) KEGG pathway enrichment of 90 candidate genes.

FIGURE 2

Identification of biomarkers associated with asparagine in T2DM. (A) PPI network analysis of 80 candidate genes; (B) Subsequent integration of four algorithmic approaches (EPC, Closeness, betweenness, MCC) for candidate feature genes; (C) Boruta algorithm plot; (D) Permutation feature importance with optimal ntree = 6 (minimum error rate); (E) 4 shared feature genes between three algorithmic outputs yielded; (F) Violin plot of PPP1CA, CTSD, PKM, TIMP1 expression; (G) ROC analysis of PPP1CA and CTSD in the training cohort (GSE15932) and the validation cohort (GSE26168); (H) Relative mRNA expression of PPP1CA and CTSD in the T2DM and the healthy control (n = 10).

FIGURE 3

GSEA of PPP1CA and CTSD in T2DM. (A) GSEA of PPP1CA in T2DM; (B) GSEA of CTSD in T2DM.

FIGURE 4

Immune microenvironment analysis of T2DM progression. (A) Hotmap of immune-infiltrating cells between T2DM and healthy control group; (B) ssGSEA of immune-infiltrating cells scores between T2DM and healthy control group; (C) Correlation analysis of immune-infiltrating cells; (D) Correlation analysis of immune-infiltrating cells with PPP1CA and CTSD.

FIGURE 5

Subcellular localization and multi-layer regulatory network analysis of PPP1CA and CTSD in T2DM. (A) Subcellular localization of PPP1CA and CTSD; (B) GeneMANIA network analysis of PPP1CA and CTSD; (C) Transcriptional regulation analysis of PPP1CA and CTSD; (D) miRNA-mRNA network analysis of PPP1CA and CTSD; (E) lncRNAs networks of PPP1CA and CTSD.

FIGURE 6

Pharmacological compounds analysis of PPP1CA and CTSD. (A) Pharmacological compounds network analysis for PPP1CA and CTSD; (B) Molecular docking of norcantharidin and PPP1CA; (C) Molecular docking of naringenin and CTSD.