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P. B. Liton, Y. Lin, C. Luna, P. Gonzalez, D. L. Epstein; Identification of Genes Differentially Expressed by Chitinase 3-Like 1 in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4859.
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© ARVO (1962-2015); The Authors (2016-present)
Chitinase 3 like 1 (Chi3L1) is an extracellular protein of unknown function previously identified by our laboratory as one of the most abundant transcripts in outflow pathway cells with high levels of expression in the aqueous humor. Our objective was to investigate the potential role of Chi3L1 in the outflow pathway through the analysis of the differential gene expression profile resulting from the over-expression and down-regulation of Chi3L1 in cultured human trabecular (HTM) meshwork cells.
To modulate the expression of Chi3L1, we constructed replication-deficient recombinant adenoviruses containing either the coding region of human Chi3L1 (AdChi3L1), or a microRNA sequence design to specifically knockdown the expression of human Chi3L1 (AdmiChi3L1). Control viruses included AdSEAP, containing the coding region of the secreted alkaline phophatase protein, and AdmiR-NC, containing a miRNA not predicted to target any known vertebrate gene. Three independent primary cultures of HTM cells were transduced with 10 pfu/cell of AdChi3L1, AdmiChi3L1, AdSEAP, or AdmiR-NC. At day three post-infection, total RNA was isolated and individually hybridized to DNA spotted arrays HO36K (Operon Human Oligo Set, Version 4.0, Total= 12 arrays). Analysis of the data was performed using Genespring GX and Ingenuity Pathway Analysis (IPA) software. Results were confirmed by qPCR and Western-blot (WB) analysis.
qPCR and WB analysis demonstrated the upregulated and downregulated expression of Chi3L1 in HTM cells transduced with AdChi3L1 and AdmiChi3L1, respectively. No significant changes in Chi3L1 expression were observed with AdSEAP and AdmiR-NC. Overexpression of Chi3L1 did not result in dramatic changes in gene expression among the different cell lines. This is probably due to the already high constitutive levels of expression of Chi3L1 in outflow pathway cells. More biologically and statistically significant results were obtained from knocking down the expression of Chi3L1, which showed 88 genes commonly upregulated and 131 genes commonly downregulated more than 1.5-fold in all the three cell lines, including MMP-1, IL-beta1, and several members of the complement cascade. IPA analysis of the dataset generated by Genespring identified cancer, cell death, inflammatory disease, and connective tissue disorders as the top biological functions affected by Chi3L1.
Our results suggest a protective role of Chi3L1 against inflammation, ECM remodeling, and cell death in the outflow pathway.
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