RNA-Seq Analysis Unraveling Novel Genes and Pathways Influencing Corneal Wound Healing
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ABSTRACT:
PROVIDER: PRJNA1081851 | ENA |
REPOSITORIES: ENA
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Project description:Transparent avascular cornea providing two third refraction to the eye. Restoration of corneal transparency and clear vision in a traumatic eye involves the action of many cytokines and signaling pathways. Out of several factors, stromal keratocytes/fibroblasts (CSFs) play a central role in corneal repair and wound healing. Post trauma, keratocytes/fibroblasts produce myofibroblasts to facilitate wound repair by synthesizing and secreting large extracellular matrix components, collagens, and alpha-smooth muscle actin stress fibers. This study aimed to perform RNASeq data analysis and pathway enrichments to gain a better understanding of gene regulation in corneal fibroblasts and myofibroblasts in corneal wound repair.
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Project description:Cornea, the outermost transparent layer of the eye, is the first line of defense against external threats. Following injury, the wound healing response is crucial to corneal repair and regeneration, yet its underlying mechanism is poorly understood. Our study was designed to investigate the role of dsRNA and its regulatory network in corneal wound healing.
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Project description:Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing. Primary cultures of human corneal epithelial cells cultivated on BSA (number of replicates: 7), Collagen type I (number of replicates: 2), Collagen type IV (number of replicates: 2), Fibronectin (number of replicates: 2), Tenascin C (number of replicates: 2) and Laminin (number of replicates: 2) matrix. Central, internal and external ring of wounded Tissue-engineered human cornea.
2015-11-25 | E-GEOD-75336 | biostudies-arrayexpress
Project description:The precise role of long non-coding RNAs (lncRNAs) as key epigenetic regulators in mediating corneal epithelial wound healing remains elusive. Here, we aim to elucidate the functional contribution of lncRNAs in regulating CEWH. We used Microarray to characterize lncRNA expression profiling during mouse CEWH. The full thickness of the corneal epithelium of C57BL/6 mice, was removed using a 0.5-mm corneal rust ring remover. Subsequently, the entire corneal epithelium was collected from both the injured and fellow eyes for RNA isolation after approximately 48 hours. Microarray analysis revealed dysregulation of numerous lncRNA candidates during CEWH. Out of the 41,655 non-coding RNAs detected by the microarray, 639 were upregulated and 1,006 were downregulated during the process of CEWH.
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Project description: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. 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. 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.
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Project description:Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing.
2015-11-25 | GSE75336 | GEO
Project description:The cornea, transparent and outermost structure of camera-type eyes, is prone to environmental challenges, but has remarkable wound healing capabilities which enables to preserve vision. The manner in which cell plasticity impacts wound healing remains to be determined. In this study, we report rapid wound closure after zebrafish corneal epithelium abrasion. Furthermore, by investigating the cellular and molecular events taking place during corneal epithelial closure, we show the induction of a bilateral response to a unilateral wound. Our transcriptomic results, together with our TGF-beta receptor inhibition experiments, demonstrate conclusively the crucial role of TGF-beta signaling in corneal wound healing. Finally, our results on Pax6 expression and bilateral wound healing, demonstrate the decisive impact of epithelial cell plasticity on the pace of healing. Altogether, our study describes terminally differentiated cell competencies in the healing of an injured cornea. These findings will enhance the translation of research on cell plasticity to organ regeneration.
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Project description: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. 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.
2013-11-09 | GSE52233 | GEO