Identifying potential pathogenesis and immune infiltration in diabetic foot ulcers using bioinformatics and in vitro analyses

Yuanyuan Xu; Jianchang Xu; Sirong Chen; Anbang Zhou; Guangjing Huang; Shidao Huang; Dianbo Yu; Biaoliang Wu

doi:10.1186/s12920-023-01741-2

Identifying potential pathogenesis and immune infiltration in diabetic foot ulcers using bioinformatics and in vitro analyses

BMC Med Genomics. 2023 Dec 1;16(1):313. doi: 10.1186/s12920-023-01741-2.

Authors

Yuanyuan Xu^{1

2}, Jianchang Xu³, Sirong Chen^{1

2}, Anbang Zhou^{1

2}, Guangjing Huang^{1

2}, Shidao Huang^{1

4}, Dianbo Yu^{1

4}, Biaoliang Wu⁵

Affiliations

¹ Graduate School, Youjiang Medical University for Nationalities, Baise, 533000, Guangxi, China.
² Department of Endocrinology, The Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, 533000, Guangxi, China.
³ The First Clinical College of Wuhan University, Wuhan, 430000, China.
⁴ Department of Orthopedics, The Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, 533000, Guangxi, China.
⁵ Department of Endocrinology, The Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, 533000, Guangxi, China. yymucun@ymun.edu.cn.

Abstract

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.

Keywords: Diabetic foot ulcers; Dual-luciferase reporter assay; Immune infiltration; Immunofluorescence staining; miRNAs-mRNA; qPCR.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Computational Biology / methods
Diabetes Mellitus*
Diabetic Foot* / genetics
Gene Expression Profiling
Humans
Luciferases / genetics
MicroRNAs* / genetics
MicroRNAs* / metabolism
RNA, Messenger / genetics
RNA, Messenger / metabolism

Substances

MicroRNAs
RNA, Messenger
Luciferases
MIRN338 microRNA, human