Identification and clinical validation of the role of anoikis-related genes in diabetic foot.

This study aims to investigate the role of anoikis-related genes in diabetic foot (DF) by utilizing bioinformatics analysis to identify key genes associated with anoikis in DF. We selected the GEO datasets GSE7014, GSE80178 and GSE68183 for the extraction and analysis of differentially expressed anoikis-related genes (DE-ARGs). GO analysis and KEGG analysis indicated that DE-ARGs in DF were primarily enriched in apoptosis, positive regulation of MAPK cascade, anoikis, focal adhesion and the PI3K-Akt signalling pathway. Based on the LASSO and SVM-RFE algorithms, we identified six characteristic genes. ROC curve analysis revealed that these six characteristic genes had an area under the curve (AUC) greater than 0.7, indicating good diagnostic efficacy. Expression analysis in the validation set revealed downregulation of CALR in DF, consistent with the training set results. GSEA results demonstrated that CALR was mainly enriched in blood vessel morphogenesis, endothelial cell migration, ECM-receptor interaction and focal adhesion. The HPA database revealed that CALR was moderately enriched in endothelial cells, and CALR was found to interact with 63 protein-coding genes. Functional analysis with DAVID suggested that CALR and associated genes were enriched in the phagosome component. CALR shows promise as a potential marker for the development and treatment of DF.

HMOX1 as a therapeutic target associated with diabetic foot ulcers based on single-cell analysis and machine learning.

Diabetic foot ulcers (DFUs) are a serious chronic complication of diabetes mellitus and a leading cause of disability and death in diabetic patients. However, current treatments remain unsatisfactory. Although macrophages are associated with DFU, their exact role in this disease remains uncertain. This study sought to detect macrophage-related genes in DFU and identify possible therapeutic targets. Single-cell datasets (GSE223964) and RNA-seq datasets (GSM68183, GSE80178, GSE134431 and GSE147890) associated with DFU were retrieved from the gene expression omnibus (GEO) database for this study. Analysis of the provided single-cell data revealed the distribution of macrophage subpopulations in the DFU. Four independent RNA-seq datasets were merged into a single DFU cohort and further analysed using bioinformatics. This included differential expression (DEG) analysis, multiple machine learning algorithms to identify biomarkers and enrichment analysis. Finally, key results were validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western bolt. Finally, the findings were validated using RT-qPCR and western blot. We obtained 802 macrophage-related genes in single-cell analysis. Differential expression analysis yielded 743 DEGs. Thirty-seven macrophage-associated DEGs were identified by cross-analysis of marker genes with macrophage-associated DEGs. Thirty-seven intersections were screened and cross-analysed using four machine learning algorithms. Finally, HMOX1 was identified as a potentially valuable biomarker. HMOX1 was significantly associated with biological pathways such as the insulin signalling pathway. The results showed that HMOX1 was significantly overexpressed in DFU samples. In conclusion, the analytical results of this study identified HMOX1 as a potentially valuable biomarker associated with macrophages in DFU. The results of our analysis improve our understanding of the mechanism of macrophage action in this disease and may be useful in developing targeted therapies for DFU.

MicroRNA-221-3p inhibits the inflammatory response of keratinocytes by regulating the DYRK1A/STAT3 signaling pathway to promote wound healing in diabetes.

Diabetic foot ulcer (DFU), a serious complication of diabetes, remains a clinical challenge. MicroRNAs affect inflammation and may have therapeutic value in DFU. Here, we find that an miR-221-3p mimic reduces the inflammatory response and increases skin wound healing rates in a mouse model of diabetes, whereas miR-221-3p knockout produced the opposite result. In human keratinocytes cells, miR-221-3p suppresses the inflammatory response induced by high glucose. The gene encoding DYRK1A is a target of miR-221-3p. High glucose increases the expression of DYRK1A, but silencing DYRK1A expression decreases high glucose-induced inflammatory cytokine release via dephosphorylation of STAT3, a substrate of DYRK1A. Application of miR-221-3p mimic to human keratinocytes cells not only decreases DYRK1A expression but also inhibits high glucose-induced production of inflammatory cytokines to promote wound healing. This molecular mechanism whereby miR-221-3p regulates inflammation through the DYRK1A/STAT3 signaling pathway suggests targets and therapeutic approaches for treating DFU.

Correlational analysis of PLIN1 with inflammation in diabetic foot ulcer wounds.

BACKGROUND: The persistent presence of inflammation is a recognized pathogenic mechanisms of diabetic foot ulcers (DFUs). We aimed to investigate the expression of PLIN1 in tissues from DFU patients and assess its potential association with inflammation-induced damage. METHODS: We performed transcriptome sequencing and correlation analysis of the foot skin from patients with or without DFUs. Additionally, we examined the correlation between PLIN1 and related inflammatory indicators by analyzing PLIN1 expression in tissue and serum samples and through high-glucose stimulation of keratinocytes (HaCaT cells). RESULTS: PLIN1 is upregulated in the tissue and serum from DFU patients. Additionally, PLIN1 shows a positive correlation with leukocytes, neutrophils, monocytes, C-reactive protein, and procalcitonin in the serum, as well as IL-1ß and TNF-α in the tissues. Experiments with Cells demonstrated that reduced expression of PLIN1 leads to significantly decreased expression of iNOS, IL-1ß, IL-6, IL-18, and TNF-α. PLIN1 may mediate wound inflammatory damage through the NF-κB signaling pathway. CONCLUSION: Our findings suggest that PLIN1 mediates the inflammatory damage in DFU, offering new prospects for the treatment of DFU.

A novel diabetic foot ulcer diagnostic model: identification and analysis of genes related to glutamine metabolism and immune infiltration.

BACKGROUND: Diabetic foot ulcer (DFU) is one of the most common and severe complications of diabetes, with vascular changes, neuropathy, and infections being the primary pathological mechanisms. Glutamine (Gln) metabolism has been found to play a crucial role in diabetes complications. This study aims to identify and validate potential Gln metabolism biomarkers associated with DFU through bioinformatics and machine learning analysis. METHODS: We downloaded two microarray datasets related to DFU patients from the Gene Expression Omnibus (GEO) database, namely GSE134431, GSE68183, and GSE80178. From the GSE134431 dataset, we obtained differentially expressed Gln-metabolism related genes (deGlnMRGs) between DFU and normal controls. We analyzed the correlation between deGlnMRGs and immune cell infiltration status. We also explored the relationship between GlnMRGs molecular clusters and immune cell infiltration status. Notably, WGCNA to identify differentially expressed genes (DEGs) within specific clusters. Additionally, we conducted GSVA to annotate enriched genes. Subsequently, we constructed and screened the best machine learning model. Finally, we validated the predictions' accuracy using a nomogram, calibration curves, decision curve analysis (DCA), and the GSE134431, GSE68183, and GSE80178 dataset. RESULTS: In both the DFU and normal control groups, we confirmed the presence of deGlnMRGs and an activated immune response. From the GSE134431 dataset, we obtained 20 deGlnMRGs, including CTPS1, NAGS, SLC7A11, GGT1, GCLM, RIMKLA, ARG2, ASL, ASNS, ASNSD1, PPAT, GLS2, GLUD1, MECP2, ASS1, PRODH, CTPS2, ALDH5A1, DGLUCY, and SLC25A12. Furthermore, two clusters were identified in DFU. Immune infiltration analysis indicated the presence of immune heterogeneity in these two clusters. Additionally, we established a Support Vector Machine (SVM) model based on 5 genes (R3HCC1, ZNF562, MFN1, DRAM1, and PTGDS), which exhibited excellent performance on the external validation datasetGSE134431, GSE68183, and GSE80178 (AUC = 0.929). CONCLUSION: This study has identified five Gln metabolism genes associated with DFU, revealing potential novel biomarkers and therapeutic targets for DFU. Additionally, the infiltration of immune-inflammatory cells plays a crucial role in the progression of DFU.

Exploring shared therapeutic targets in diabetic cardiomyopathy and diabetic foot ulcers through bioinformatics analysis.

Advanced diabetic cardiomyopathy (DCM) patients are often accompanied by severe peripheral artery disease. For patients with DCM combined with diabetic foot ulcer (DFU), there are currently no good therapeutic targets and drugs. Here, we investigated the underlying network of molecular actions associated with the occurrence of these two complications. The datasets were downloaded from the Gene Expression Omnibus (GEO) database. We performed enrichment and protein-protein interaction analyses, and screened for hub genes. Construct transcription factors (TFs) and microRNAs regulatory networks for validated hub genes. Finally, drug prediction and molecular docking verification were performed. We identified 299 common differentially expressed genes (DEGs), many of which were involved in inflammation and lipid metabolism. 6 DEGs were identified as hub genes (PPARG, JUN, SLC2A1, CD4, SCARB1 and SERPINE1). These 6 hub genes were associated with inflammation and immune response. We identified 31 common TFs and 2 key miRNAs closely related to hub genes. Interestingly, our study suggested that fenofibrate, a lipid-lowering medication, holds promise as a potential treatment for DCM combined with DFU due to its stable binding to the identified hub genes. Here, we revealed a network involves a common target for DCM and DFU. Understanding these networks and hub genes is pivotal for advancing our comprehension of the multifaceted complications of diabetes and facilitating the development of future therapeutic interventions.

Understanding molecular mechanisms and miRNA-based targets in diabetes foot ulcers.

In today's culture, obesity and overweight are serious issues that have an impact on how quickly diabetes develops and how it causes complications. For the development of more effective therapies, it is crucial to understand the molecular mechanisms underlying the chronic problems of diabetes. The most prominent effects of diabetes are microvascular abnormalities such as retinopathy, nephropathy, and neuropathy, especially diabetes foot ulcers, as well as macrovascular abnormalities such as heart disease and atherosclerosis. MicroRNAs (miRNAs), which are highly conserved endogenous short non-coding RNA molecules, have been implicated in several physiological functions recently, including the earliest stages of the disease. By binding to particular messenger RNAs (mRNAs), which cause mRNA degradation, translation inhibition, or even gene activation, it primarily regulates posttranscriptional gene expression. These molecules exhibit considerable potential as diagnostic biomarkers for disease and are interesting treatment targets. This review will provide an overview of the latest findings on the key functions that miRNAs role in diabetes and its complications, with an emphasis on the various stages of diabetic wound healing.

Mapping cellular senescence networks in human diabetic foot ulcers.

Cellular senescence, a cell fate defined by irreversible cell cycle arrest, has been observed to contribute to chronic age-related conditions including non-healing wounds, such as diabetic foot ulcers. However, the role of cellular senescence in the pathogenesis of diabetic foot ulcers remains unclear. To examine the contribution of senescent phenotypes to these chronic wounds, differential gene and network analyses were performed on publicly available bulk RNA sequencing of whole skin biopsies of wound edge diabetic foot ulcers and uninvolved diabetic foot skin. Wald tests with Benjamini-Hochberg correction were used to evaluate differential gene expression. Results showed that cellular senescence markers, CDKN1A, CXCL8, IGFBP2, IL1A, MMP10, SERPINE1, and TGFA, were upregulated, while TP53 was downregulated in diabetic foot ulcers compared to uninvolved diabetic foot skin. NetDecoder was then used to identify and compare context-specific protein-protein interaction networks using known cellular senescence markers as pathway sources. The diabetic foot ulcer protein-protein interaction network demonstrated significant perturbations with decreased inhibitory interactions and increased senescence markers compared to uninvolved diabetic foot skin. Indeed, TP53 (p53) and CDKN1A (p21) appeared to be key regulators in diabetic foot ulcer formation. These findings suggest that cellular senescence is an important mediator of diabetic foot ulcer pathogenesis.

Stem cell therapy with CRISPR/Cas9-mediated MALAT1 delivery modulates miR-142 and rescues wound healing in rats with age-associated diabetic foot ulcers.

BACKGROUND: Diabetic foot ulcer (DFU) is a serious diabetes complication, significantly impacting the quality of life, particularly in the elderly. Age-associated DFUs pose additional challenges due to impaired healing mechanisms. Our study aims to explore the role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) as a miR-142 sponge in repairing diabetic rat foot ulcer tissue under age-associated diabetes, offering a new theoretical basis and therapeutic target for preventing and treating diabetic vascular disease in the elderly. METHODS: Using qPCR, we analyzed MALAT1 and miR-142 expression in EPCs and hUC-MSCs. Targetscan predicted potential interaction targets for MALAT1 and miR-142, confirmed by dual luciferase reporter gene assay. An age-associated diabetic rat model was established using Streptozotocin (STZ) injection. Hypoxia, apoptosis, and angiogenesis-related proteins were assessed through Western Blot. In vitro, miR-142 inhibition and MALAT1 overexpression promoted foot ulcer healing in diabetic rats. RESULTS: MALAT1 acted as a miR-142 sponge, downregulated in hUC-MSCs under high glucose, relevant to age-associated diabetic foot ulcers. MiR-142 negatively regulated SIRT1 and Nrf2. In vitro experiments demonstrated potential significance for age-related DFU treatment. CONCLUSIONS: MALAT1 in human umbilical cord mesenchymal stem cells expedited foot ulcer healing in diabetic rats, particularly in age-associated diabetes, through miR-142 sponge activity. These findings offer insights for novel therapeutic strategies targeting elderly diabetic foot ulcers, emphasizing exogenous stem cell transplantation's potential in effective DFU treatment for the elderly.

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

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.

Unraveling shared risk factors for diabetic foot ulcer: a comprehensive Mendelian randomization analysis.

INTRODUCTION: Diabetic foot ulcer (DFU) stands as a severe diabetic lower extremity complication, characterized by high amputation rates, mortality, and economic burden. We propose using Mendelian randomization studies to explore shared and distinct risk factors for diabetic lower extremity complications. RESEARCH DESIGN AND METHODS: We selected uncorrelated genetic variants associated with 85 phenotypes in five categories at the genome-wide significance level as instrumental variables. Genetic associations with DFU, diabetic polyneuropathy (DPN), and diabetic peripheral artery disease (DPAD) were obtained from the FinnGen and UK Biobank studies. RESULTS: Body mass index (BMI) emerged as the only significant risk factor for DPAD, DPN, and DFU, independent of type 2 diabetes, fasting glucose, fasting insulin, and HbA1c. Educational attainment stood out as the sole significant protective factor against DPAD, DPN, and DFU. Glycemic traits below the type 2 diabetes diagnosis threshold showed associations with DPAD and DPN. While smoking history exhibited suggestive associations with DFU, indicators of poor nutrition, particularly total protein, mean corpuscular hemoglobin, and mean corpuscular volume, may also signal potential DFU occurrence. CONCLUSIONS: Enhanced glycemic control and foot care are essential for the diabetic population with high BMI, limited education, smoking history, and indicators of poor nutrition. By focusing on these specific risk factors, healthcare interventions can be better tailored to prevent and manage DFU effectively.

Identification of potential diagnostic biomarkers and immune infiltration features in diabetic foot ulcer by bioinformatics analysis and validation.

Diabetic foot ulcer (DFU) is the most serious and costly chronic complication that may lead to disability and even death in patients suffering from diabetes mellitus (DM). However, the clinical diagnosis and prognosis of DFU is inadequate. There is still a lack of effective biomarkers for its early diagnosis. We obtained the circRNA expression dataset GSE114248 and mRNA expression dataset GSE80178 from the GEO. R software was used to identify the differentially expressed circRNAs (DECs). The mRNAs associated with DFU were identified by a random forest algorithm and intersected with mRNAs predicted by circRNAs. Then, the circRNA-miRNA-mRNA network was established and the hub genes were screened using GO semantic similarity and were validated by the GSE199939 dataset. Meanwhile, the expression level of the biomarkers was verified by RT-PCR assays and immunohistochemistry. Finally, GSEA was conducted to determine differential immune cell infiltration and the immunological cells' relationships with hub genes. We identified three hub genes including KIAA1109, ENPP5, and NRP1 that might play an important role in DFU. ROC curve results also showed a good performance of these three genes in the validation dataset. Furthermore, RT-PCR assays and immunohistochemistry confirmed the results above. Immune infiltration analysis indicated that DFU had a significant increase in Neutrophils. Moreover, three hub genes were closely correlated with a variety of inflammatory cells. KIAA1109, ENPP5, and NRP1 are key hub genes of DFU. They might play an important role in the development of DFU and could be potential biomarkers in DFU.

Complementary Gene Therapy after Revascularization with the Saphenous Vein in Diabetic Foot Syndrome.

Diabetic foot syndrome (DFS) is one of the most serious macroangiopathic complications of diabetes. The primary treatment option is revascularization, but complementary therapies are still being sought. The study group consisted of 18 patients diagnosed with ischemic ulcerative and necrotic lesions in DFS. Patients underwent revascularization procedures and, due to unsatisfactory healing of the lesions, were randomly allocated to two groups: a group in which bicistronic VEGF165/HGF plasmid was administered and a control group in which saline placebo was administered. Before gene therapy administration and after 7, 30, 90, and 180 days, color duplex ultrasonography (CDU) was performed, the ankle-brachial index (ABI) and transcutaneous oxygen pressure (TcPO2) were measured, and DFS changes were described and documented photographically. In the gene therapy group, four out of eight patients (50%) healed their DFS lesions before 12 weeks. During this time, the ABI increased by an average of 0.25 and TcPO2 by 30.4 mmHg. In the control group, healing of the lesions by week 12 occurred in six out of nine patients (66.67%), and the ABI increased by an average of 0.14 and TcPO2 by 27.1 mmHg. One major amputation occurred in each group. Gene therapy may be an attractive option for complementary treatment in DFS.

Exploring the pathogenesis of osteomyelitis accompanied by diabetic foot ulcers using microarray data analysis.

Although numerous studies have shown distinctive similarities between osteomyelitis and diabetic foot ulcers (DFU), the common pathogenesis of both is not fully understood. The current research focuses on an in-depth study of the molecular and pathway mechanisms involved in the complication of these 2 diseases. We downloaded clinical information on osteomyelitis (GSE30119) and DFU (GSE29221) from the GEO database, along with gene expression matrices. Differentially expressed genes (DEGs) among normal individuals and patients with osteomyelitis; normal individuals and patients with DFU were identified by R software, and thus common DEGs were confirmed. We then analyzed these differential genes, including the functional pathway analysis, protein-protein interaction (PPI), modules and hub genes establishment, and transcription factor regulatory networks. We identified 109 common DEGs (46 up-regulated and 63 down-regulated genes) for subsequent analysis. The results of PPI network and the functional pathway analysis revealed the importance of immune response and inflammatory response in both diseases. Among them, chemokines and cytokines were found to be closely related to both osteomyelitis and DFU. In addition, the tumor necrosis factor (TNF) pathway and Staphylococcus aureus infection were found to have more significant roles too. The 12 most essential key genes were later screened by cytoHubba, including matrix metalloproteinases (MMP) 1, MMP3, MMP9, IL8, C-X-C chemokine receptor (CXCR) 2, C-X-C motif chemokine ligand (CXCL) 9, CXCL10, CXCL13, FCGR3B, IL1B, LCN2, S100A12. CXCL10, and MMP1 were validated using the least absolute shrinkage and selection operator (LASSO) and support vector machine-recursive feature elimination (SVM-RFE) algorithms. Osteomyelitis and DFU share similar molecular and pathway mechanisms. These common key genes and pathways may provide new directions toward the future study of osteomyelitis and DFU.

Adipose-derived stem cell exosome NFIC improves diabetic foot ulcers by regulating miR-204-3p/HIPK2.

BACKGROUND: Diabetic foot ulcers (DFU) are a serious complication of diabetes that lead to significant morbidity and mortality. Recent studies reported that exosomes secreted by human adipose tissue-derived mesenchymal stem cells (ADSCs) might alleviate DFU development. However, the molecular mechanism of ADSCs-derived exosomes in DFU is far from being addressed. METHODS: Human umbilical vein endothelial cells (HUVECs) were induced by high-glucose (HG), which were treated with exosomes derived from nuclear factor I/C (NFIC)-modified ADSCs. MicroRNA-204-3p (miR-204-3p), homeodomain-interacting protein kinase 2 (HIPK2), and NFIC were determined using real-time quantitative polymerase chain reaction. Cell proliferation, apoptosis, migration, and angiogenesis were assessed using cell counting kit-8, 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, wound healing, and tube formation assays. Binding between miR-204-3p and NFIC or HIPK2 was predicted using bioinformatics tools and validated using a dual-luciferase reporter assay. HIPK2, NFIC, CD81, and CD63 protein levels were measured using western blot. Exosomes were identified by a transmission electron microscope and nanoparticle tracking analysis. RESULTS: miR-204-3p and NFIC were reduced, and HIPK2 was enhanced in DFU patients and HG-treated HUVECs. miR-204-3p overexpression might abolish HG-mediated HUVEC proliferation, apoptosis, migration, and angiogenesis in vitro. Furthermore, HIPK2 acted as a target of miR-204-3p. Meanwhile, NFIC was an upstream transcription factor that might bind to the miR-204-3p promoter and improve its expression. NFIC-exosome from ADSCs might regulate HG-triggered HUVEC injury through miR-204-3p-dependent inhibition of HIPK2. CONCLUSION: Exosomal NFIC silencing-loaded ADSC sheet modulates miR-204-3p/HIPK2 axis to suppress HG-induced HUVEC proliferation, migration, and angiogenesis, providing a stem cell-based treatment strategy for DFU.

LncRNA SNHG16 Knockdown Promotes Diabetic Foot Ulcer Wound Healing via Sponging MiR-31-5p.

Diabetic foot ulcers are caused by nerve abnormalities and vascular lesions in the distal lower limbs of diabetic patients. However, the causes of diabetic foot ulcers are diverse and the treatment process is complex. Therefore, understanding the pathogenesis of diabetic foot ulcers through lncRNA and formulating effective means are the key to the cure of patients. Tissues were collected from 76 diabetic foot ulcer patients and 50 non-diabetic patients undergoing traumatic amputation. Human dermal fibroblasts (HDFs) were induced by high glucose to obtain diabetic foot ulcer cell model. The lncRNA SNHG16 (SNHG16) and miR-31-5p expression in tissues and cells was detected by real-time quantitative reverse transcription PCR (RT-qPCR). Cell Counting Kit-8 (CCK-8) and Transwell assays were used to evaluate the biological behavior of the cells, and the association between SNHG16 and miR-31-5p was explored by luciferase reporting assay. SNHG16 was distinctly expressed in diabetic foot ulcer tissue samples, while miR-31-5p was decreased. In vitro cell function assays confirmed that the proliferation level was inhibited in the constructed diabetic foot ulcer cell model (HG group), as was the migration and invasion ability. After transfection with silencing SNHG16, the biological behavior of the cells was promoted. Mechanistically, SNHG16 sponge miR-31-5p regulated disease progression. Recovery experiments revealed that miR-31-5p inhibitor counteracted the effect of silencing SNHG16 on cell viability. SNHG16 knockdown may regulate the biological function of cells by targeting miR-31-5p to promote wound healing and ameliorate the condition of diabetic foot ulcer patients.

Circ_072697 knockdown promotes advanced glycation end products-induced cell proliferation and migration in HaCaT cells via miR-3150a-3p/KDM2A axis.

OBJECTIVE: Diabetes foot ulcer (DFU) is a serious complication of diabetes, which can lead to significant mortality and amputation rate. Our previous study found circ_072697 was highly expressed in DFU tissues, but the regulatory mechanism of circ_072697 in DFU remains unclear. METHODS: The relative expressions of circ_072697, miR-3150a-3p, and KDM2A in DFU patients or advanced glycation end products (AGEs)-treated HaCaT cells (used as DFU cell model) were determined by using qRT-PCR. Cell proliferation and migration abilities were determined by using CCK-8 and Transwell assays. The interaction between miR-3150a-3p with circ_072697 or KDM2A were verified by RNA immunoprecipitation (RIP) and dual-luciferase reporter assays. Furthermore, the protein expression of genes involved in MAPK signaling pathway was detected by western blot. RESULTS: The expression of circ_072697 was significantly upregulated in DFU tissues, while the expression of miR-3150a-3p was downregulated. Circ_072697 knockdown promoted the proliferation and migration of AGEs-treated HaCaT cells. miR-3150a-3p was confirmed as a target of circ_072697 and its inhibitor reversed the promotion effects of circ_072697 knockdown on biological behavior of cells. In addition, KDM2A was considered as a target of miR-3150a-3p and it was highly expressed in DFU samples. Importantly, circ_072697 could regulate KDM2A expression through sponging miR-3150a-3p, and this axis had effect on the MAPK signaling pathway. CONCLUSIONS: Overall, circ_072697 regulated the biological behaviors of keratinocytes in DFU via miR-3150a-3p/KDM2A axis and MAPK signaling pathway, revealing a new insight into the pathogenesis and potential therapeutic targets of DFU.

Transcriptomic identification of genes expressed in invasive S. aureus diabetic foot ulcer infection.

Introduction: Infection in diabetic foot ulcers (DFUs) is one of the major complications associated with patients with diabetes. Staphylococcus aureus is the most common offending pathogen in patients with infected DFU. Previous studies have suggested the application of species-specific antibodies against S. aureus for diagnosis and monitoring treatment response. Early and accurate identification of the main pathogen is critical for management of DFU infection. Understanding the host immune response against species-specific infection may facilitate diagnosis and may suggest potential intervention options to promote healing infected DFUs. We sought to investigate evolving host transcriptome associated with surgical treatment of S. aureus- infected DFU. Methods: This study compared the transcriptome profile of 21 patients with S. aureus- infected DFU who underwent initial foot salvage therapy with irrigation and debridement followed by intravenous antibiotic therapy. Blood samples were collected at the recruitment (0 weeks) and 8 weeks after therapy to isolate peripheral blood mononuclear cells (PBMCs). We analyzed the PBMC expression of transcriptomes at two different time points (0 versus 8 weeks). Subjects were further divided into two groups at 8 weeks: healed (n = 17, 80.95%) versus non-healed (n = 4, 19.05%) based on the wound healing status. DESeq2 differential gene analysis was performed. Results and discussion: An increased expression of IGHG1, IGHG2, IGHG3, IGLV3-21, and IGLV6-57 was noted during active infection at 0 weeks compared with that at 8 weeks. Lysine- and arginine-rich histones (HIST1H2AJ, HIST1H2AL, HIST1H2BM, HIST1H3B, and HIST1H3G) were upregulated at the initial phase of active infection at 0 weeks. CD177 and RRM2 were also upregulated at the initial phase of active infection (0 weeks) compared with that at 8 weeks of follow-up. Genes of heat shock protein members (HSPA1A, HSPE1, and HSP90B1) were high in not healed patients compared with that in healed patients 8 weeks after therapy. The outcome of our study suggests that the identification of genes evolution based on a transcriptomic profiling could be a useful tool for diagnosing infection and assessing severity and host immune response to therapies.

Upregulation of circ_0080968 in diabetic foot ulcer inhibits wound healing via repressing the migration and promoting proliferation of keratinocytes.

BACKGROUND: Diabetic foot ulcer (DFU) is a serious chronic complication of diabetes mellitus whose pathogenesis remains unclear. Circular RNA (circRNA) refers to a group of covalently closed non-coding RNAs that are reported to be dysregulated in patients with DFU. However, the mechanism whereby dysregulation in circRNAs contributes to DFU remains unclear. In this study, we investigated the role of dysregulated circRNAs in DFU. MATERIALS AND METHODS: A gene expression dataset was downloaded from the Gene Expression Omnibus portal and analyzed by the limma package of R. The levels of 24 upregulated circRNAs were detected in two independent cohorts by RT-qPCR. Interactions between miRNAs and circRNAs were predicted through bioinformatics and confirmed using a dual luciferase assay. The circularity and subcellular localization of circRNA-080968 was examined by northern blotting after digestion with RNase-R and in situ hybridization. Cell migration and proliferation were examined using Transwell and MTT assays. The apoptotic cells were detected by flow cytometry. RESULTS: The level of circRNA-080968 was upregulated in DFU tissues compared to that of non-DFU samples and normal human wounds. CircRNA-080968 was mainly localized in the cytoplasm and its overexpression inhibited the migration and promoted the proliferation of keratinocytes. MiR-326 and miR-766-3p were identified to interact with and be negatively correlated with circRNA-080968 levels. Increased glucose upregulated circRNA-080968, and its overexpression accelerated the degradation of both miR-326 and miR-766-3p. Reduced levels of miR-326 and miR-766-3p upregulated the expression of several genes controlling cell adhesion and proliferation which are related to the pathogenesis of DFU. CONCLUSIONS: The upregulation of circRNA-080968 in DFU induced the degradation of miR-326 and miR-766-3p, which further repressed the migration and increased the proliferation of keratinocytes.

RIG-I contributes to keratinocyte proliferation and wound repair by inducing TIMP-1 expression through NF-κB signaling pathway.

Epithelial keratinocyte proliferation is an essential element of wound repair, and chronic wound conditions, such as diabetic foot, are characterized by aberrant re-epithelialization. In this study, we examined the functional role of retinoic acid inducible-gene I (RIG-I), a key regulator of epidermal keratinocyte proliferation, in promoting TIMP-1 expression. We found that RIG-I is overexpressed in keratinocytes of skin injury and underexpressed in skin wound sites of diabetic foot and streptozotocin-induced diabetic mice. Moreover, mice lacking RIG-I developed an aggravated phenotype when subjected to skin injury. Mechanistically, RIG-I promoted keratinocyte proliferation and wound repair by inducing TIMP-1 via the NF-κB signaling pathway. Indeed, recombinant TIMP-1 directly accelerated HaCaT cell proliferation in vitro and promoted wound healing in Ddx58-/- and diabetic mice in vivo. In summary, we demonstrated that RIG-I is a crucial factor that mediates epidermal keratinocyte proliferation and may be a potential biomarker for skin injury severity, thus making it an attractive locally therapeutic target for the treatment of chronic wounds such as diabetic foot.