Simultaneous Integrative Analysis of MicroRNA(miR) & Gene Expression to Contrast Optic Nerve Head(ONH) Responses to Optic Nerve Transection(ONT) with a Controlled Elevation of IOP(CEI)

Abstract

Purpose : To characterize and contrast the early biological ONH responses between ONT & CEI by integrating miR Array and RNA-Seq data.

Methods : Rats underwent unilateral optic nerve transection (ONT)(n=8) and were sacrificed 3 days later. Another group, also sacrificed at 3 days, underwent unilateral exposure to an 8-hour CEI at 60mmHg(n=8). Total RNA was extracted from all ONHs and from naïve controls(n=10).

MiR expression was studied with Array Cards & differential expression identified by ΔΔCt after global normalization. Statistical comparison was performed using a 2-tailed t-test. Within the same ONH, gene expression was studied by RNA-Seq. Data were mapped to the UC Santa Cruz rat genome with unique reads normalized by the trimmed mean of M-values. A generalized linear model with likelihood ratio tests identified differentially expressed genes(compared to controls). Genes & miRs with adjusted p<0.05 stratified for fold changes >±2 were considered significant.

Computationally-predicted gene targets of dysregulated miRs were integrated with RNA-Seq data to identify anti-correlated gene-miR pairs. Functional enrichment studies were performed by computing overlaps of the integrated data with hallmark gene sets using the Molecular Signatures Database.

Results : 3 down & 15 upregulated miRs, compared to controls, were identified after ONT(Fig 1). A mean of 44 anti-correlated pairs corresponding to increased gene activity(gene↑miR↓) were noted. These included cell proliferation, inflammatory responses, apoptosis and IL-2/Stat5 signaling. Reduced biological activity(5 pairs,gene↓miR↑) post-ONT was consistent with reduced axonal function and cell differentiation.

4 down & 8 upregulated miRs were identified after CEI(Fig 2),with a mean of 5(gene↑miR↓) & 3(gene↓miR↑) anti-correlated pairs. Reduced axonal function was seen in CEI ONHs.

The 9-fold difference in anti-correlated gene-miR pairs corresponding to increased activity(gene↑miR↓) suggests more active responses develop post-ONT than after CEI.

Conclusions : Functional integration of anti-correlated miR-mRNA pairs suggests that ONT and CEI create different biological responses within the ONH. Understanding the dynamic nature of these paired responses as injury develops following CEI will advance our understanding of the complex pathophysiology of glaucomatous optic neuropathy.

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