Abstract
Purpose :
LOXL1-AS1 is a long non-coding RNA associated with pseudoexfoliation glaucoma (PEXG). PEXG is characterized by the disrupted regulation of aqueous outflow and increased intraocular pressure. LOXL1-AS1 responds to mechanical stress, controls transcription, and patients with PEXG display fibrillary deposits in the eye; thus, we hypothesized that LOXL1-AS1 regulates genes involved in extracellular matrix (ECM) remodeling, cell adhesion, and mechanotransduction pathways in outflow cells.
Methods :
LOXL1-AS1 was knocked down in primary cultures of human trabecular meshwork (TM) and Schlemm’s Canal (SC) cells (4 strains each) using targeted Ad-shRNA in cells, and in human lens epithelial cells (HLE-B3) cells by transfection with a targeted siRNA. Knockdown (KD) verification and ECM gene expression were determined using qPCR or RNAseq. Genes with significant expression differences were further investigated using western blots, as were the mechanotransduction hub proteins, FAK, MAPK, pMLC, and AKT.
Results :
Only data from experiments with >50% of LOXL1-AS1 knocked down was analyzed. In HLE-B3 cells with LOXL1-AS1 KD, 7 genes were significantly altered (p<.05). Thrombospondin 2 and Tenascin C were upregulated by 2-fold (p<.05) while TIMP1 decreased by 50% (p<.05). After KD of LOXL1-AS1 in TM cells, the expression of 7 ECM genes were significantly altered (p<.05). Of these, col11α1 showed a 50% decrease in expression and integrinβ3’s expression doubled (p<.05). Genes affected by LOXL1-AS1 KD in TM were Col6α6 (4-fold increase, n=3, p=.04) and Col3α1 (1.5 fold increase, n=3, p=.001). When LOXL1-AS1 was KD in SC cells, 13 genes showed significantly altered RNA expression. Of these, integrinα2 was upregulated by 2.5-fold and lamininλ1 decreased by 50% (p<.05), but protein validation did not show a significant change. Of the mechanotransduction proteins analyzed, the phosphorylation of AKT was significantly elevated in SC cells (n=4, p =.008), and trending toward elevation in TM cells (n=4, p=.056).
Conclusions :
LOXL1-AS1 regulates many genes in a cell type-dependent manner. For outflow cells, LOXL1-AS1 controls the expression of key ECM components and AKT, a central mechanotransduction protein. Thus, polymorphisms in LOXL1-AS1 may contribute to fibrillary deposits, cell signaling, and/or ECM homeostasis in the conventional outflow pathway of the eye of PEXG patients.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.