Identifying potential pathogenesis and immune infiltration in diabetic foot ulcers using bioinformatics and in vitro
Yuanyuan Xu1,2, Jianchang Xu3, Sirong Chen1,2
1Graduate School, Youjiang Medical University for Nationalities, Baise, 533000, Guangxi, China.
Background:
Diabetic foot ulcers (DFU) are among the fastest-growing diseases worldwide. Recent evidence has emphasized the critical role of microRNA (miRNA)-mRNA networks in various chronic wounds, including DFU. In this study, we aimed to clarify the miRNA-mRNA axes associated with the occurrence of DFU.
Methods:
Expression profiles of miRNAs and mRNAs were extracted from the Gene Expression Omnibus. Differentially expressed genes and differentially expressed miRNAs were identified, and miRNA-mRNA regulatory axes were constructed through integrated bioinformatics analyses. We validated the miRNA-mRNA axes using quantitative real-time PCR (qPCR) and dual-luciferase reporter assays. We conducted an immune infiltration analysis and confirmed the bioinformatics results using immunofluorescence staining. Single-sample gene set enrichment analysis (ssGSEA) was used to analyze the metabolic mechanisms.
Results:
miR-182-5p-CHL1/MITF and miR-338-3p-NOVA1 interactions were identified using in silico analysis. The qPCR results showed apparent dysregulation of these miRNA-mRNA axes in DFU. The dual-luciferase reporter assay confirmed that miR-182-5p targeted CHL1 and MITF, and miR-338-3p targeted NOVA1. We conducted an immune infiltration analysis and observed that key genes correlated with decreased infiltration of M1 macrophages and resting mast cells in DFU. Immunofluorescence staining verified the co-localization of CHL1 and tryptase, while MITF and CD68 showed weak positive correlations. Metabolic pathways related to these three genes were identified using ssGSEA.
Conclusions:
In summary, the miR-182-5p-CHL1/MITF and miR-338-3p-NOVA1 pathway interactions and decreased infiltration of M1 macrophages and resting mast cells may provide novel clues to the pathogenesis of DFU.
Trial Registration:
The clinical trial included in this study was registered in the Chinese Clinical Trial Registry ( ChiCTR2200066660 ) on December 13, 2022.
Insights
This study identifies key microRNA-mRNA interactions, specifically miR-182-5p-CHL1/MITF and miR-338-3p-NOVA1, involved in diabetic foot ulcers (DFU). These findings link altered immune cell infiltration to DFU pathogenesis.
Area of Science:
- Molecular Biology
- Genomics
- Immunology
Background:
- Diabetic foot ulcers (DFU) represent a growing global health challenge.
- MicroRNA (miRNA)-mRNA regulatory networks are increasingly recognized for their role in chronic wound development, including DFU.
- Understanding these complex networks is crucial for elucidating DFU pathogenesis.
Purpose of the Study:
- To identify and characterize specific miRNA-mRNA axes implicated in the development of diabetic foot ulcers.
- To investigate the relationship between these miRNA-mRNA interactions and immune cell infiltration in DFU.
- To explore the metabolic pathways associated with key regulatory genes in DFU.
Main Methods:
- Bioinformatic analysis of miRNA and mRNA expression profiles from public databases (Gene Expression Omnibus).
- Identification of differentially expressed miRNAs and mRNAs to construct regulatory axes.
- Validation of miRNA-mRNA interactions using quantitative real-time PCR (qPCR) and dual-luciferase reporter assays.
- Immune infiltration analysis and immunofluorescence staining to assess immune cell presence and localization.
- Single-sample gene set enrichment analysis (ssGSEA) for metabolic pathway investigation.
Main Results:
- In silico analysis revealed potential interactions: miR-182-5p targeting CHL1 and MITF, and miR-338-3p targeting NOVA1.
- qPCR confirmed significant dysregulation of these miRNA-mRNA axes in DFU samples.
- Dual-luciferase assays validated the direct targeting of CHL1, MITF, and NOVA1 by their respective miRNAs.
- Immune infiltration analysis indicated a correlation between key genes and reduced M1 macrophage and resting mast cell infiltration in DFU.
- Immunofluorescence confirmed co-localization patterns for CHL1/tryptase and MITF/CD68, with ssGSEA identifying relevant metabolic pathways.
Conclusions:
- The identified miR-182-5p-CHL1/MITF and miR-338-3p-NOVA1 pathway interactions are significantly associated with diabetic foot ulcer development.
- Decreased infiltration of M1 macrophages and resting mast cells may play a role in DFU pathogenesis.
- These findings offer novel insights into the molecular mechanisms underlying DFU and suggest potential therapeutic targets.
