Differential miRNA Expression in Response to Mechanical Stress in Human Trabecular Meshwork Cells

Abstract

Purpose : miRNAs have been shown to play important roles in regulating trabecular meshwork (TM) function and aqueous outflow. We aimed to identify differentially expressed miRNAs in human TM (HTM) cells in response to long-term mechanical stress.

Methods : Six strains of primary HTM cells were isolated from cadaveric eyes with no history of eye disease and cultured under serum-starved condition in the presence or absence of 15% mechanical stretch, 1 cycle/second, for 24 hours using the computer controlled Flexcell Unit. Total RNA was isolated using Ambion mirVana miRNA Isolation kit. Expression of 800 human miRNAs was profiled using NanoString nCounter Human miRNA Assay kits. After quality check and normalization, differentially expressed miRNAs in the stretched condition were identified using Bioconductor Limma package. Droplet digital PCR was used to validate the differential expression of selected miRNAs.

Results : 373 miRNAs were detectably expressed in the cultured HTM cells. Of these miRNAs, 74 miRNAs (10 down- and 64 up-regulated) were differentially expressed in stretched cells (p≤0.05). The top 5 most significantly expressed miRNAs were miR-32-5p, miR-4286, miR-135-5p, miR-136-5p, miR-93-5p (p value= 1.2x10^-5, 1.3x10^-5, 4.5x10^-5, 7x10^-5, 2x10^-4 respectively). The miRNAs with the greatest fold change in response to stretch included miR-4286, miR-32-5p, miR-136-5p, miR-93-5p, miR-135-5p, miR-21-5p, miR-19b-3p with log2 fold change ranging from 1.3 to 0.98. Consistent with previous reports, miR-100-5p, miR-27a-3p, miR-27b-3p, miR-24-3p, miR-16-5p, let-7f-5p and let-7i-5p were significantly upregulated in stretched versus control HTM cells. Ingenuity Pathway Analysis showed that these miRNAs are mostly associated with cellular functions related to proliferation, development, cell cycle regulation and cellular movement. Remarkably, the highly expressed miRNAs are predicted regulators of CDKN2A, a glaucoma-related gene shown to be upregulated in a rat glaucoma model.

Conclusions : Our miRNA profiling suggests a potential role of miRNAs in homeostatic responses of HTM cells to in vivo physiological mechanical stress. Further characterization of these miRNAs may advance our understanding of HTM-mediated regulation of aqueous outflow resistance and generate possible therapeutic targets to regulate intraocular pressure.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.