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Differentially expressed wound healing-related microRNAs in the human diabetic cornea.

ABSTRACT: MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by in situ hybridization (ISH) in independent samples. HCEC were transfected with human pre-miR™miRNA precursors (h-miR) or their inhibitors (antagomirs) using Lipofectamine 2000. Confluent transfected cultures were scratch-wounded with P200 pipette tip. Wound closure was monitored by digital photography. Expression of signaling proteins was detected by immunostaining and Western blot. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing.

SUBMITTER: Funari VA

PROVIDER: S-EPMC3869828 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Differentially expressed wound healing-related microRNAs in the human diabetic cornea.

Funari Vincent A VA Winkler Michael M Brown Jordan J Dimitrijevich Slobodan D SD Ljubimov Alexander V AV Saghizadeh Mehrnoosh M

PloS one 20131220 12

MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybri ...[more]

PMID: 24376808

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Project description:ObjectiveWound healing is a complex process involving the coordinated interaction of various genes and molecularpathways. The study aimed to uncover novel therapeutic targets, biomarkers and candidate genes for drug developmentto improve successful wound repair interventions.Materials and Methods: This study is a network-meta analysis study. Nine wound healing microarray datasets obtainedfrom the Gene Expression Omnibus (GEO) database were used for this study. Differentially expressed genes (DEGs)were described using the Limma package and shared genes were used as input for weighted gene co-expressionnetwork analysis. The Gene Ontology analysis was performed using the EnrichR web server, and construction of aprotein-protein interaction (PPI) network was achieved by the STRING and Cytoscape.Results: A total of 424 DEGs were determined. A co-expression network was constructed using 7692 shared genesbetween nine data sets, resulting in the identification of seven modules. Among these modules, those with the top 20genes of up and down-regulation were selected. The top down-regulated genes, including TJP1, SEC61A1, PLEK,ATP5B, PDIA6, PIK3R1, SRGN, SDC2, and RBBP7, and the top up-regulated genes including RPS27A, EEF1A1,HNRNPA1, CTNNB1, POLR2A, CFL1, CSNk1E, HSPD1, FN1, and AURKB, which can potentially serve as therapeutictargets were identified. The KEGG pathway analysis found that the majority of the genes are enriched in the "Wntsignaling pathway".Conclusion: In our study of nine wound healing microarray datasets, we identified DEGs and co-expressed modulesusing WGCNA. These genes are involved in important cellular processes such as transcription, translation, and posttranslationalmodifications. We found nine down-regulated genes and ten up-regulated genes, which could serve aspotential therapeutic targets for further experimental validation. Targeting pathways related to protein synthesis and celladhesion and migration may enhance wound healing, but additional experimental validation is needed to confirm theeffectiveness and safety of targeted interventions.

Project description:IntroductionWound healing is very important for the maintenance of immune barrier integrity, which has attracted wide attention in past 10 years. However, no studies on the regulation of cuproptosis in wound healing have been reported.MethodsIn this study, the skin injury model was constructed in Gnxi goats, and the function, regulatory network and hub genes of the skin before and after the injury were comprehensively analyzed by transcriptomics.ResultsThe results showed that there were 1,438 differentially expressed genes (DEGs), genes up-regulated by 545 and genes down-regulated by 893, which were detected by comparing day 0 and day 5 posttraumatic skin. Based on GO-KEGG analysis, DEGs that were up-regulated tended to be enriched in lysosome, phagosome, and leukocyte transendothelial migration pathways, while down-regulated DEGs were significantly enriched in adrenergic signaling in cardiomyocytes and calcium signaling pathway. There were 166 overlapped genes (DE-CUGs) between DEGs and cuproptosis-related genes, with 72 up-regulated DE-CUGs and 94 down-regulated DE-CUGs. GOKEGG analysis showed that up-regulated DE-CUGs were significantly enriched in ferroptosis, leukocyte transendothelial migration and lysosome pathways, while down-regulated DE-CUGs were significantly enriched in Apelin signaling pathway and tyrosine metabolism pathways. By constructing and analyzing of protein-protein interaction (PPI) networks of DEGs and DE-CUGs, 10 hub DEGs (ENSCHIG00000020079, PLK1, AURKA, ASPM, CENPE, KIF20A, CCNB2, KIF2C, PRC1 and KIF4A) and 10 hub DE-CUGs (MMP2, TIMP1, MMP9, MMP14, TIMP3, MMP1, EDN1, GCAT, SARDH, and DCT) were obtained, respectively.DiscussionThis study revealed the hub genes and important wound healing pathways in Ganxi goats, and identified the correlation between wound healing and cuproptosis for the first time, and found that MMP2, TIMP1, MMP9, and EDN1 were the core genes associated. This study enriched the transcriptome data of wound healing in Ganxi goats and expanded the research direction of cuproptosis.

Dataset Information

Differentially expressed wound healing-related microRNAs in the human diabetic cornea.

Publications

Differentially expressed wound healing-related microRNAs in the human diabetic cornea.

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