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Inas F Aboobakar, David Corcoran, William Marshall Johnson, R Rand Allingham, W Daniel Stamer, Michael A Hauser; Knockdown of the lncRNA LOXL1-AS1 and the protein-coding gene LOXL1 alters expression of genes and pathways implicated in exfoliation syndrome pathogenesis. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5622. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Exfoliation syndrome (XFS) is a systemic disorder of the extracellular matrix (ECM) affecting ~60 million people. Exfoliation glaucoma (XFG) is the most common ocular manifestation. Coding and non-coding variants in the LOXL1 locus increase risk for developing XFG, though the functional effects remain poorly characterized. The goal of this work was to identify the genes and pathways regulated by the two genes in this locus: the non-coding lncRNA LOXL1-AS1 and the protein coding gene LOXL1.
Immortalized human lens epithelial cells (HLE-B3) were transfected with an siRNA targeting either the LOXL1-AS1 lncRNA (3 siRNA probes tested), LOXL1 (2 probes tested) or a negative control siRNA. Each probe was tested in triplicate. Total RNA was extracted from cells after 48 hours and RNA-seq analysis was performed using 50bp single end reads. The DESeq2 Bioconductor package was used to identify genes differentially expressed with LOXL1-AS1 or LOXL1 knockdown (KD) compared to the negative control (p<0.05, >2-fold change). Gene set enrichment analysis (GSEA) was used to identify differentially expressed pathways.
KD of LOXL1-AS1 led to differential expression of 632 genes (425 protein-coding, 207 non-coding), while LOXL1 KD led to differential expression of 254 genes (107 protein-coding, 147 non-coding). Differentially expressed genes included various exfoliation material and ECM constituents, matrix metalloproteinases, members of the TGF-β superfamily, calcium signaling genes and genes involved in aqueous humor dynamics (Table 1). Pathway analysis identified enrichment of genes involved in UV response, proteasome complex formation, ECM constituents and downstream targets of TGF-β1 with LOXL1-AS1 KD. Pathways differentially expressed with LOXL1 KD included response to UV light, actin cytoskeleton assembly and ECM constituents.
Knockdown of LOXL1-AS1 or LOXL1 affects expression of genes and pathways implicated in XFS pathobiology, including ECM constituents, the TGF-β1 pathway and response to XFS-associated stressors such as UV light. Altered expression of LOXL1-AS1 and/or LOXL1 due to genetic variants or environmental factors may induce changes in ECM homeostasis and impair responses to cell stressors, contributing to the development of the ocular and systemic manifestations of this ECM-related disease.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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