Abstract
Purpose :
Elevated intraocular pressure contributes to glaucomatous optic nerve degeneration by inducing biomechanical stress at the optic nerve head (ONH), especially in the lamina cribrosa (LC). ONH astrocytes (ONHA) in the LC respond to biomechanical signals through extracellular matrix (ECM) remodeling activities, promoting tissue fibrosis and damage to retinal ganglion cell axons. MicroRNAs (miRNA, miR) may play a role in regulating these activities. Previous miRNA profiling by our lab suggested the miRNA 146 family may be upregulated in stretched ONHA. In this study, we quantified the expression of miR-146b-5p and miR-146b-3p in stretched ONHA and examined potential downstream targets.
Methods :
Primary human ONHA strains (n=3) were exposed to 0-12% cyclic stretch for 24h. RNA samples and conditioned medium samples were collected. Primer assays for mir-146b-5p and miR-146b-3p were used to determine expression changes. TargetScan was used to identify potential mRNA targets of mir-146b. Protein levels for candidate targets were examined using western blotting of concentrated conditioned medium. ECM protein modifications were assessed by probing for post-translational modifications on fibronectin immunoprecipitated from conditioned medium samples.
Results :
In stretched ONHA, miR-146b-5p expression increased 1.55-fold (p=0.002) and miR-146b-3p increased 1.48-fold (p=0.003). Predicted mRNA targets for both microRNAs included ECM proteins and proteins involved in ECM dynamics. In particular, miR-146b-3p is predicted to target fibronectin (FN) and transglutaminase 2 (TGM2). No changes in total levels of secreted FN were observed. Extracellular TGM2 was increased 2.57-fold (p=0.007) in stretched ONHA. Immunoblotting of immunoprecipitated fibronectin demonstrated a 53.54% (p=0.007) increase in primary amine transamidated fibronectin.
Conclusions :
Cyclic stretch increased ONHA expression of mir-146b. Extracellular levels of TGM2 were increased with stretch, and increased fibronectin post-translational modifications suggest an increased activity of TGM2 – an activity often associated with increased ECM protein crosslinking and fibrosis. miR-146b-3p is predicted to target TGM2, suggesting that it could negatively regulate ECM crosslinking in stretched ONHA. Our next experiments will be focused on validating miR-146b targeting of TGM2 and characterizing the activity of TGM2 in stretched ONHA.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.