Dataset Information

Expression profile analysis to identify potential gene changes induced by dexamethasone in the trabecular meshwork.

ABSTRACT:

Aim

To investigate potential gene changes in trabecular meshwork (TM) induced by dexamethasone (DEX) in steroid-induced glaucoma (SIG).

Methods

The expression data of 24 cases from a public functional genomics data were sorted to identify the mechanisms of action of DEX on the TM. The relationships of the differentially expressed genes (DEGs) were enriched using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. In addition, the hub genes were screened by the Search Tool for the Retrieval of Interacting Genes Database (STRING) and Cytoscape tools. Finally, human TM cells (HTMCs) were treated with DEX to preliminarily explore the function of hub genes.

Results

Totally 47 DEGs, including 21 downregulated and 26 upregulated genes were identified. The primary enriched results of the DEGs consisted of inflammatory response, extracellular matrix (ECM), negative regulation of cell proliferation, TNF signalling pathway and the regulation of tryptophan channels by inflammatory mediators. Subsequently, pro-melanin-enriched hormone (PMCH) and Bradykinin B1 receptor (BDKRB1) were screened as hub genes. It is verified in GSE37474 data set. Western blot and quantitative real-time polymerase chain reaction (qPCR) results showed that protein and RNA expression levels of BDKRB1 were significantly decreased after DEX treatment, while PMCH was not significantly changed.

Conclusion

BDKRB1 may be a key gene involved in SIG onset, providing a suitable therapeutic target for improving the prognosis of SIG patients.

SUBMITTER: Wei M

PROVIDER: S-EPMC9358186 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

Expression profile analysis to identify potential gene changes induced by dexamethasone in the trabecular meshwork.

Wei Miao M Chen Lu-Ming LM Huang Ze-Yu ZY Zhang Guo-Wei GW Guan Huai-Jin HJ Ji Min M

International journal of ophthalmology 20220818 8

<h4>Aim</h4>To investigate potential gene changes in trabecular meshwork (TM) induced by dexamethasone (DEX) in steroid-induced glaucoma (SIG).<h4>Methods</h4>The expression data of 24 cases from a public functional genomics data were sorted to identify the mechanisms of action of DEX on the TM. The relationships of the differentially expressed genes (DEGs) were enriched using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. In addition, the hub genes were screened ...[more]

PMID: 36017046

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Project description:Changes in the actin cytoskeleton, especially the formation of cross-linked actin networks (CLANs) are thought to contribute to the increased intraocular pressure observed in primary open-angle and steroid-induced glaucoma. To better understand the effects of glucocorticoids, we employed a shotgun method to analyze global changes in the cytoskeleton and integrin signaling pathways following dexamethasone (DEX) treatment of human trabecular meshwork (HTM) cells. RNA and cell lysates were obtained from HTM cells incubated with or without DEX. Changes in protein expression were determined by mass spectrometry (MS) following differential centrifugation of cell lysates to enrich for low-abundance cytoskeletal and signaling proteins, proteolytic digestion, and a titanium dioxide column to enrich for phosphopeptides. Results were validated by Western blots. Changes in RNA levels were determined with gene arrays and RT-PCR. Overall, MS identified 318 cytoskeleton associated proteins. Five of these proteins (PDLIM1, FGFR1OP, leiomodin-1, ZO-2 and LRP16A) were only detected in DEX-treated cells by MS. However, only PDLIM1 showed a statistically significant increase at the RNA level. Other proteins with differences at both the RNA and protein levels included ?3 integrin, caveolin-1, Borg2, raftlin1, PI-3 kinase regulatory subunit ?, transgelin, and filamin B. By immunofluorescence microscopy filamin B and PDLIM1 showed enhanced expression in human trabecular meshwork cells, but only PDLIM1 demonstrated significant localization within CLANs. Finally, MS showed that some of the cytoskeleton proteins (Borg2, leiomodin-1, LRP16A, raftlin1 and CKAP4) contained phosphorylated residues. This study suggests that DEX affects the expression of cytoskeleton proteins at the transcriptional and translational level and shows that a combined genomic and proteomic approach can be used for rapid analysis of proteins in the TM. It also shows that DEX altered the expression of components (PDLIM1 and ?3 integrins) involved in CLAN formation and provides new findings into the effects of glucocorticoids on the cytoskeleton.

Project description:BackgroundTrabecular meshwork and Schlemm's canal are the tissues appointed to modulate the aqueous humour outflow from the anterior chamber. The impairment of their functions drives to an intraocular pressure increase. The selective laser trabeculoplasty is a laser therapy of the trabecular meshwork able to decrease intraocular pressure. The exact response mechanism to this treatment has not been clearly delineated yet. The herein presented study is aimed at studying the gene expression changes induced in trabecular meshwork cells by selective laser trabeculoplasty (SLT) in order to better understand the mechanisms subtending its efficacy.Methodology/principal findingsPrimary human trabecular meshwork cells cultured in fibroblast medium underwent selective laser trabeculoplasty treatment. RNA was extracted from a pool of cells 30 minutes after treatment while the remaining cells were further cultured and RNA was extracted respectively 2 and 6 hours after treatment. Control cells stored in incubator in absence of SLT treatment were used as reference samples. Gene expression was evaluated by hybridization on miRNA-microarray and laser scanner analysis. Scanning electron microscopic examination was performed on 2 Trabecular meshwork samples after SLT at 4(th) and 6(th) hour from treatment. On the whole, selective laser trabeculoplasty modulates in trabecular meshwork the expression of genes involved in cell motility, intercellular connections, extracellular matrix production, protein repair, DNA repair, membrane repair, reactive oxygen species production, glutamate toxicity, antioxidant activities, and inflammation.Conclusions/significanceSLT did not induce any phenotypic alteration in TM samples. TM is a complex tissue possessing a great variety of function pivotal for the active regulation of aqueous humour outflow from the anterior chamber. SLT is able to modulate these functions at the postgenomic molecular level without inducing damage either at molecular or phenotypic levels.

Dataset Information

Expression profile analysis to identify potential gene changes induced by dexamethasone in the trabecular meshwork.

Aim

Methods

Results

Conclusion

Publications

Expression profile analysis to identify potential gene changes induced by dexamethasone in the trabecular meshwork.

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