Association of miR-182 with High Tension Glaucoma

Abstract

Purpose : miRNAs may play important roles in many eye disorders including glaucoma. Our integrative study examined the role of miR-182 in primary open-angle glaucoma (POAG).

Methods : Our study integrated genetic association and miRNA expression profiling. First, using the NEIGHBORHOOD cohort (3853 cases/33495 controls), we performed a case-control association analysis with 85 common variants located in 76 miRNA genes. Stratified association analyses were performed with high tension glaucoma (HTG, maximum IOP>21 mmHg, 1868 cases) and normal tension glaucoma (NTG, maximum IOP≤21 mmHg, 725 cases). Second, next generation miRNA sequencing with Illumina MiSeq was performed with total RNAs extracted from normal human eye tissue (2 ciliary body, 2 cornea, 2 retina, 4 trabecular meshwork (TM), and 1 aqueous humor (AH)). Third, Realtime PCR and droplet digital PCR (ddPCR) were used to validate the expression of miR-182 in human ocular tissues and aqueous humor. Fourth, exosomes isolated from the conditioned media of primary human TM cells were characterized using nanoparticle tracking analysis for size distribution and quantity while exosomal RNA was profiled using miRNA sequencing. Fifth, using ddPCR we compared miR-182 expression in AH from 5 HTG cases vs. 5 controls.

Results : First, we identified a significant association between rs76481776 and POAG (odd ratio 1.23, p=0.00019). This variant is located within the precursor of the MIR182 gene. Further analyses indicated significant association with HTG (p=0.004), but not NTG (p>0.05). The risk allele T has been associated with 30% higher expression of mature miR-182 in vitro. Second, miRNA sequencing indicated miR-182 expression in all examined ocular tissues. Third, the relative expression of miR-182 in these tissues was validated using realtime PCR and ddPCR. Fourth, miR-182 was expressed in TM derived exosomes, which have a mean diameter of 100nm. Fifth, miR-182 expression was 2-fold higher in HTG AH than non-glaucoma AH (p=0.03).

Conclusions : It has previously been reported that miR-182 is up-regulated (7-9 fold) with stress-induced premature senescence in TM cells. Overexpression of miR-182 may contribute to the senescent response through regulating its target genes in DNA repair and cell cycle. One of these target genes is CHEK2, whose variants have been associated with HTG. Our study is the first to suggest the association of elevated miR-182 expression with HTG.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.